Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface.

soma:schamberger-phbec-ali-001

Primary human bronchial epithelial cells differentiated at the air-liquid interface

CILIARY:schamberger-ciliated-cells-d28

Acetylated-tubulin-positive ciliated cell percentage after 28 days of ALI differentiation with chronic CSE

Immunofluorescence quantification of acTUB-positive ciliated cells over 28 days of pHBEC differentiation at ALI, with or without chronic CSE. The ciliated cell fraction rose over time in all cultures and was unchanged by CSE up to day 21; at day 28 chronic CSE strikingly reduced it in a concentration-dependent manner. The paper reports the day 28 percentages in prose as approximations: controls approximately 50-55% (about 53%), CSE-exposed cultures only about 30%. The discussion pools the two CSE concentrations when stating the ~30% figure, so the treated output record is tied to a pooled chronic-CSE condition rather than to a single dose. No spread is given for these prose values.

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Evidence

FOXJ:schamberger-foxj1-mrna-28d

FOXJ1 mRNA and FOXJ1 target gene transcripts under chronic CSE (no change)

NULL RESULT. qRT-PCR for FOXJ1 across 28 days of ALI differentiation with or without chronic CSE (2.5% or 5%). FOXJ1 mRNA rose greatly over time in control conditions, correlating with ciliated cell numbers, but chronic CSE produced no alteration in FOXJ1 mRNA at any time point. The reported FOXJ1 target genes DNAI1, DNALI1, SPAG6 and TEKT1 were likewise unchanged. The paper reports these data graphically only, so no expression values are recorded. This is the basis for the authors' conclusion that the loss of ciliated cells is FOXJ1-independent.

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GENEXPR:schamberger-teer-28d

Transepithelial electrical resistance during pHBEC differentiation under chronic CSE

TEER was followed over 28 days of ALI differentiation with or without 2.5% or 5% CSE. Chronic CSE significantly decreased TEER from day 14 on, in a concentration-dependent manner, so CSE impaired establishment of the bronchial barrier in differentiating basal cells. The paper shows TEER graphically only (Figure 1c) and states no numeric resistances in the text, so the output records carry no values. The concentrations used were first shown to be non-toxic by LDH assay over the first 72 hours.

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GENEXPR:schamberger-cyp1a1-mrna-28d

CYP1A1 transcript induction by CSE during pHBEC differentiation

qRT-PCR of CYP1A1, the canonical aryl hydrocarbon receptor target, over 28 days of ALI differentiation. Both 2.5% and 5% CSE highly induced CYP1A1 transcript at all time points relative to control, which the authors use to confirm CSE potency. CYP1A1 baseline expression declined over the course of differentiation, with only limited detection at day 21 and day 28. Fold inductions are shown graphically only (Figure 1d), so no expression values are recorded.

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Evidence

GENEXPR:schamberger-actub-protein-28d

Acetylated tubulin protein levels under chronic CSE

Western blot of acetylated tubulin (acTUB), the ciliated cell marker, across 28 days of ALI differentiation. acTUB protein tracked FOXJ1 mRNA and ciliated cell numbers over time in control cultures. Both CSE concentrations decreased acTUB from day 14 after air-lift, the time point at which first motile cilia became detectable by immunofluorescence. Blot densitometry for acTUB is not tabulated in the paper, so no values are recorded. The reduction in acTUB is the protein-level counterpart of the cell-number change, and the authors link it to the reduced cilia length reported in smokers.

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Evidence

GENEXPR:schamberger-cc10-positive-cells-d28

CC10-positive Clara (club) cell percentage under chronic CSE

Immunofluorescence quantification of CC10-positive Clara cells over 28 days of ALI differentiation. The Clara cell fraction peaked at about 20% at day 14 in all conditions. Control and 2.5% CSE cultures then dropped gradually to about 10% by day 28, whereas 5% CSE cultures did not decline between day 14 and day 28 and held about 17% at day 28 (p < 0.01). Percentages are stated in prose as approximations and no spread is given for them. NOTE ON THE KEY EVENT: the shared key event is "Club cell marker loss" (decreased), but this paper reports club cell NUMBER going UP under CSE, so the evidence here is recorded as REFUTE. The companion assay on intracellular CC10 protein supports the same key event.

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GENEXPR:schamberger-cc10-protein-28d

Intracellular and secreted CC10 protein under chronic CSE

Western blot of CC10 in cell lysates and in cell supernatants across 28 days of ALI differentiation. Continuous CSE significantly decreased intracellular CC10 protein in a concentration-dependent manner from day 14 after air-lift, while secreted CC10 increased in the supernatants of CSE-exposed cells. CC10 mRNA was unaffected at every time point, so the change is post-transcriptional. Blots are shown without tabulated densitometry, so no values are recorded. This loss of the intracellular club cell marker is what supports the club cell marker loss key event, even though the club cell COUNT rose (companion assay).

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GENEXPR:schamberger-krt14-protein-d28

KRT5 and KRT14 basal cell protein levels under chronic CSE

Western blot with densitometric quantification (mean +/- SEM, four independent differentiations, relative to GAPDH) of the basal cell keratins KRT5 and KRT14. In control cultures both proteins fell significantly over the course of differentiation (p < 0.01 vs day 7). Continuous CSE produced a trend for increased KRT5 and KRT14 protein from day 21 after air-lift, which reached significance for KRT14 at day 28. TP63, KRT5 and KRT14 mRNA did not change significantly over differentiation, so this too is a post-transcriptional change. The paper reports the densitometry graphically only, so no values are recorded. The retained basal cell marker expression is the authors' evidence that basal cell differentiation is impaired by CSE.

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GENEXPR:schamberger-ivl-mrna-28d

Involucrin (IVL) transcript levels under chronic CSE

qRT-PCR of involucrin, a marker for squamous metaplasia, over 28 days of ALI differentiation. IVL transcript declined over time in control conditions but was significantly elevated at day 7 and day 28 in the presence of CSE. Values are shown graphically only (Figure 4d), so no expression values are recorded. IVL is the only differentiation marker in the paper whose TRANSCRIPT level CSE changed.

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GENEXPR:schamberger-differentiated-cells-d28

Total marker-positive (differentiated) cell percentage at day 28 under chronic CSE

The fraction of cells carrying any of the three differentiation markers (acTUB, CC10, MUC5AC) at day 28, quantified from the same immunofluorescence images. On average 73.6% of cells were differentiated in control conditions, dropping to 57.9% under 2.5% CSE and 63.9% under 5% CSE — an overall reduction in differentiated cells, which the authors read together with the raised basal cell markers as impaired basal cell differentiation. The paper itself cautions that these percentages must be viewed with caution, because MUC5AC/CC10 double-positive cells exist and are therefore counted under more than one marker; no spread or p-value is given for them.

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GOBLET:schamberger-muc5ac-cells-d28

MUC5AC-positive goblet cell numbers after 28 days of ALI differentiation with chronic CSE

Goblet cells were quantified by positivity for MUC5AC. In control cultures goblet cells increased up to day 21 and declined afterwards; 5% CSE significantly increased MUC5AC-positive cells at day 28. The paper states the effect size only as "a 7% increase in goblet cell numbers in fully differentiated pHBECs" and gives no absolute goblet cell percentages, so the output records carry no percentages and the stated increase is recorded in the response comparison below. The authors caution that the fractions must be viewed with caution because MUC5AC/CC10 double-positive cells exist.

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GOBLET:schamberger-muc5ac-muc5b-mrna-28d

MUC5AC and MUC5B transcript levels under chronic CSE (no change)

NULL RESULT. qRT-PCR of MUC5AC and MUC5B across 28 days of ALI differentiation. Goblet cell numbers in control conditions were well mirrored by both MUC5AC and MUC5B mRNA, but in the presence of CSE the transcript levels were largely unchanged — the goblet cell increase is therefore not transcriptionally driven. Values are shown graphically only, so the output records carry none.

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Evidence

COMP:schamberger-goblet-cells-d28

Increase in goblet cell numbers under CSE in fully differentiated pHBECs

The only change vs control that the paper states numerically rather than showing graphically. Reported in the discussion as "a 7% increase in goblet cell numbers in fully differentiated pHBECs" — the paper does not say whether this is 7 percentage points or a 7% relative increase, and gives no absolute goblet cell percentages to disambiguate it, so it is recorded as a percent change exactly as stated. Statistical support comes from the results section, which reports the day 28 increase under 5% CSE as significant but gives no exact p-value.

Statistical test: repeated measures two-way ANOVA with Bonferroni post-tests versus the non-treated control

KER:schamberger-ciliogenesis-to-mcc

Altered ciliogenesis leads to impaired mucociliary clearance

The paper's own bridge from its in vitro result to the clinical phenotype: the authors state that the reduction in ciliated cell number they measured, together with a lower ciliary beat frequency, likely contributes to the reduced mucociliary clearance seen in smokers and COPD patients. Stated as a likelihood and not measured in this study — no mucociliary clearance assay was performed — so the support is weak and the quantitative understanding low.

PROTOCOL:schamberger-if-quantification-001

Indirect immunofluorescence staining and cell-fraction quantification on transwell membranes

pHBECs were stained on the transwell membrane and the different cell fractions quantified as described previously. Cell-type-specific markers were acetylated tubulin (acTUB) for ciliated cells, CC10 for Clara cells and MUC5AC for goblet cells, with DAPI as counterstain. 12 images per group were analysed, from 3 independent differentiations.

PROTOCOL:schamberger-qrtpcr-001

qRT-PCR of differentiation marker transcripts

Transcript levels were measured by qRT-PCR at day 7, 14, 21 and 28 of ALI differentiation. Relative transcript abundance is expressed as ddCt = [dCt(gene of interest, condition NT, day 7)] - [dCt(gene of interest, condition X, day X)] with dCt = Ct(gene of interest) - Ct(reference). For non-treated cells at day 21 or day 28, when CYP1A1 transcript levels were below detection level, Ct = 40 was used for statistical analysis.

PROTOCOL:schamberger-western-001

Western blot of differentiation markers with densitometric quantification

Western blot analysis of protein extracts from pHBECs differentiated for 7, 14, 21 or 28 days, non-treated or treated with 2.5% or 5% CSE. Blots were probed for acTUB, CC10, KRT5, KRT14 and GAPDH as loading control; secreted CC10 was blotted from the cell supernatants. Protein levels of KRT5 and KRT14 were quantified via densitometry analysis using Image Lab software, relative to GAPDH, as mean +/- SEM of four independent differentiations.

PROTOCOL:schamberger-teer-001

Transepithelial electrical resistance (TEER) measurement

TEER development of differentiating pHBECs was monitored with a Millicell-ERS-2 volt-ohm-meter and an STX01 chopstick electrode. 500 ul pre-warmed HBSS was added apically and equilibrated for 10 minutes, then a triplicate measurement was performed for every well; 6 to 12 individual wells were analysed per experiment and time point. Values were blank-corrected and multiplied by the effective membrane area (1.12 cm2 for 12-well transwell inserts).