CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization

Abstract

Background: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca²⁺ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored.

Methods: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca²⁺ dynamics and decoders to elucidate the pathway from receptor to phenotype.

Findings: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca²⁺ influx and prolonged channel closed state. This shifts the Ca²⁺ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca²⁺ dynamic changes. Ca²⁺ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation.

Interpretation: CHRFAM7A introduces an additional, human specific, layer to Ca²⁺ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis.

Funding: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Keywords: Actin cytoskeleton; Adaptation to the mechanical properties; CHRFAM7A; Ca(2+) signalling; Human immune diversity; Translational significance.

Fig. 1

Electrophysiology and Ca²⁺dynamics in UB068 (null), UB068_CHRFAM7A (direct) and UB068_CHRFAM7AΔ2bp (inverted) iPSC derived isogenic monocytes. a. Whole-cell recordings on automated high-throughput patch-clamping system on iPSC derived isogenic monocytes. Application of ACh (60 mM) and co-application of PNU120596 with 60 mM ACh with increasing concentration of PNU120596. b. PNU120596 and ACh response fold change in iPSC derived isogenic monocytes. Represented as Mean ± 95% CI (N = 5, two-way ANOVA (Cell Line and [PNU]) and Bonferroni correction for multiple testing). c. Representative fluorescent images, ROI and d. Spontaneous Ca²⁺ oscillation traces of iPSC derived isogenic monocytes. e. Cumulative density curves of Ca²⁺ peak characteristics in UB068 (black), UB068_CHRFAM7A (red) and UB068_CHRFAM7AΔ2bp (blue) monocytes. Operator independent analysis of single cell Ca²⁺ tracings was performed in MatLab. (A amplitude, FWHM full width at half maximum amplitude, f frequency). (N = 3, 50–100 peaks per cell line, two-sample KS test and Bonferroni correction for multiple testing). f. Cumulative density plots of CaBBI inferred kinetic parameters of the spontaneous Ca²⁺ dynamic curves in UB068 (black), UB068_CHRFAM7A (red) and UB068_CHRFAM7AΔ2bp (blue) monocytes. (ER pump Vmax, Ca²⁺ reuptake into the ER by SERCA, InsPR k, IP3-induced efflux from the ER, ER leak k, background efflux from the ER, IP3 t, the lifetime of IP3, IP3 (production) Vmax, activity of phospholipase C, PM influx, (constant) Ca²⁺ current across the PM into the cytosol and PM pump Vmax, evacuation of Ca²⁺ into the extracellular space by PMCA). g. The maximum distance between curves (D) and -log10p of two-sample KS test depicted in the adapted volcano plot (null-CHRFAM7A (orange) and null-CHRFAM7AΔ2bp (blue) comparison). (N = 3, 100 cells per cell line, two-sample KS test and Bonferroni correction for multiple testing). h. Proposed model of CHRFAM7A effect on Ca²⁺ dynamics (created with BioRender.com).

Fig. 2

Characterization of the α7/CHRFAM7A nAChR. a. Pharmacological modulation of the cytoplasmic Ca²⁺ pool measured by change in Ca²⁺ signal before and after treatment with agonist acetylcholine (ACh), antagonist BGT, SERCA inhibitor TG, PLC inhibitor U73122, and IP3 receptor antagonist Xesto. Represented as Mean ± 95% CI (50–100 cells, Wilcoxon signed-rank test and Bonferroni correction for multiple testing). b. Agonist and antagonist effect on amplitude in UB068 and UB068_CHRFAM7A isogenic monocytes depicted as cumulative density curves (75–100 cells per cell line, two-sample KS test and Bonferroni correction for multiple testing). c. Maximum distance between curves (D) and -log10p of two-sample KS test depicted in the adapted volcano plot (null (gray), CHRFAM7A (orange)) of Ca²⁺ dynamics changes to BGT treatment in human primary monocytes (1147 noncarrier (black) and 2407 carrier (orange) peaks analysed from 15 donors) (FWHM full width at half maximum amplitude). d. Effect of ER pool depletion (TG) and PLC inhibitor U73122, and IP3 receptor antagonist Xesto on Ca²⁺ dynamics depicted as cumulative density curves. (N = 3, 50–100 peaks per cell line, two-sample KS test and Bonferroni correction for multiple testing). e. Representative confocal images of UB068 and UB068_CHRFAM7A isogenic macrophages stained with the α7/CHRFAM7A nAChR antibody (green) and ER marker KDEL (red); colocalization (orange). Scale bar, 10 μm. f. Mander's coefficient of colocalization (M1) of ER and α7/CHRFAM7A nAChR signal calculated in ImageJ. Represented as Mean ± SD (N = 3, 25–50 cells, independent two-tailed t-test). g. Representative single cell directionality montage (3 min sample) of Fourier analysis in UB068 and UB068_CHRFAM7A isogenic monocytes. Null (UB068) demonstrating multifocal Ca²⁺ flickers and CHRFAM7A (UB068_CHRFAM7A) showing monofocal, organized, robust Ca²⁺ signal originating from the ER. h. Cumulative distribution curves of Fourier dispersion parameter in UB068 (black) and UB068_CHRFAM7A (orange) isogenic monocytes. (N = 3, 1800 images per cell line, Two-sample KS statistics, Bonferroni corrected p-values. For visualization of the distribution plots data is truncated at 95th percentile). i. Cumulative distribution curves of Fourier dispersion parameter in noncarrier (black) and carrier (orange) primary human monocytes from 15 donors (5NC, 10C). (10 random, active cells form each donor representing 600 images, Two-sample KS statistics, Bonferroni corrected p-values. For visualization of the distribution plots data is truncated at 95th percentile).

Fig. 3

CHRFAM7A associated cytoskeletal remodelling and preferred membrane structure. a. Fluorescent phalloidin microscopy images of UB068 and UB068_CHRFAM7A isogenic monocytes plated on polyacrylamide hydrogels (0.15%) demonstrating filopodia in null and lamellipodia in CHRFAM7A monocytes. Scale bar, 50 μm. b. Operator independent morphological analysis (Fiji/Image J) of UB068 and UB068_CHRFAM7A isogenic monocytes. Cumulative density curves demonstrate shift in circularity and perimeter between null (black) and CHRFAM7A (orange) monocytes (Two-sample KS statistics, Bonferroni corrected p-values). c. Representative fluorescent phalloidin microscopy images of primary human monocytes from noncarrier and carrier donors. Scale bar, 50 μm d. Operator independent morphological analysis of primary human monocytes (10NC black, 20C orange) demonstrates similar shift in morphology as seen in the iPSC derived monocytes. Carrier monocytes have larger perimeter and are less circular compared to noncarrier monocytes (Two-sample KS statistics, p-values for CHRFAM7A and null comparison after Bonferroni multiple testing correction). e. G-LISA assay demonstrating activity of small GTPases CDC42, Rac1 and RhoA in UB068 (black) and UB068_CHRFAM7A (orange) non-treated isogenic monocytes (top panel). Data are presented as mean ± SD (Independent two-tailed t-test used to compare difference in CDC42, Rac1 and RhoA activity in monocytes plated on polyacrylamide hydrogels (0.15%) compared to floating control monocytes (N = 5)). G-LISA assay of small GTPases CDC42, Rac1 and RhoA in UB068 (black) and UB068_CHRFAM7A (orange) BGT treated (middle panel), and TG treated (bottom panel) isogenic monocytes. Data are presented as mean ± SD (Independent two-tailed t-test, change in CDC42, Rac1 and RhoA activity before and after drug treatment (N = 2)). f. Pharmacological modulation of preferred membrane structure in UB068 and UB068_CHRFAM7A isogenic monocytes by BGT and TG treatment. BGT treatment of UB068 and UB068_CHRFAM7A isogenic monocytes on 0.15% polyacrylamide hydrogels confirm α7 nAChR dependence by inhibition of the preferred membrane structure: inhibition of filopodia (III) and inhibition of lamellipodia (V). TG, a SERCA inhibitor that depletes ER Ca²⁺ reservoir and reduces amplitude of Ca²⁺ dynamics, switches filopodia (I) to lamellipodia (V) in UB068 monocytes. Scale bar, 20 μm.

Fig. 4

Adaptation to tissue stiffness. a. Schematic depicting tissue stiffness measured as elastic modulus in kPa modelled by various concentrations of polyacrylamide hydrogels indicated in gold. Tissue stiffness in examples of physiological (top) and pathological (bottom) conditions (based on data from animal models) are shown. b. EVOS FL fluorescent images (objective 20×) of phalloidin stained UB068 and UB068_CHRFAM7A isogenic monocytes on a series of fibronectin-coated hydrogels from 0.03 through 0.6 corresponding to a range of elastic modulus from 2 to 30 kPa. Genotype specific preferred membrane structure is sustained at each stiffness; CHRFAM7A monocytes demonstrate adaptive morphology over the stiffness gradient. Scale bar, 20 μm. c. Operator independent morphological analysis of UB068 (black) and UB068_CHRFAM7A (orange) isogenic monocytes plated on the hydrogel gradient. UB068_CHRFAM7A (orange graph) monocytes show circularity and perimeter adaptation in opposite direction from low to high stiffness while UB068 (black graph) shape is constant over the hydrogel gradient (black). Represented as cumulative density curves (Two-sample KS statistics, Bonferroni corrected p-values)). d. Morphological adaptation of UB068 (black) and UB068_CHRFAM7A (orange) isogenic monocytes between 0.03 and 0.15% polyacrylamide hydrogel are depicted in the volcano plot (Two-sample KS statistics, maximum distance between curves (D) versus -log(p)). e. Pharmacological modulation with BGT and TG on low (0.03) stiffness hydrogel indicates that low stiffness adaptation is TG (Ca²⁺) dependent in both UB068 (black) and UB068_CHRFAM7A (orange) isogenic monocytes, albeit larger effect in UB068_CHRFAM7A (Two-sample KS statistics, Bonferroni corrected p-values)). f. On high (0.6) stiffness hydrogel, BGT blocks adaptation of UB068_CHRFAM7A (orange) monocytes indicating that high stiffness adaptation is BGT (α7/CHRFAM7A nAChR) dependent. One-dimensional cumulative distribution curves of morphological characteristics are depicted. (c, e, f: N = 3–7, 250–350 cells per genotype, Two-sample KS statistics, Bonferroni corrected p-values). g. Atomic Force microscopy (AFM). Cells were seeded on fibronectin-coated low (0.03%), intermediate (0.15%), or high (0.6%) stiffness hydrogels for 1 h. AFM was performed to measure intracellular stiffness. The stiffness data was summarized as scatter plot. (N = 3, 30 cells per condition, bars show mean ± 95% CI. Statistical tests were performed by independent two-tailed t-tests, Bonferroni corrected p-values.).

Fig. 5

Cellular adhesion, motility, and phagocytosis. a. Representative confocal images demonstrating differences in focal adhesion between UB068 and UB068_CHRFAM7A derived macrophages. Note the monolateral distribution of the cellular adhesion proteins in the null macrophages versus bilateral – in the direct. Podosomes are visualized by actin/vinculin or actin/VASP staining. Scale bar, 50 μm. b. Quantification of cell adhesion in the isogenic monocytes plated on laminin (upper panel) and Geltrex (lower panel). Spectrophotometric quantification of crystal violet stained cells represented as mean ± SD (N = 3, independent two-tailed t-tests, Bonferroni corrected p-values). c. Migration assay on UB068 and UB068_CHRFAM7A isogenic monocytes plated on transwell inserts with and without chemoattractant CXCL12 in lower chamber for 16 h. Spectrophotometric quantification of crystal violet stained cells in lower chamber are depicted as mean ± SD (N = 3, independent two-tailed t-test). d. Invasion assay on UB068 and UB068_CHRFAM7A isogenic monocytes plated on 8 mg/ml Matrigel coated transwell inserts with and without chemoattractant CXCL12 for 16 h. Spectrophotometric quantification of crystal blue stained cells in lower chamber (N = 3–5, depicted mean ± SD, independent two-tailed t-test). e. Migration through transwell inserts of primary human monocytes from 12 donors in 24 independent experiments. Box plots depict relative absorption of crystal violet stained cells in lower chamber in Noncarrier (black) and Carrier (orange) primary human monocytes (independent two-tailed t-test). f. Matrigel coated transwell invasion assay of primary human monocytes from 9 donors in 18 independent experiments (Box plots depict relative absorption of crystal violet stained cells in lower chamber). g. Pharmacological modulation of migration of UB068 isogenic monocytes with small GTPase inhibitors. Monocytes were plated with or without CDC42 (ML 141), RhoA (Rhosin) and Rac1(EHT 1862) inhibitors. Spectrophotometric quantification of crystal violet stained cells in lower chamber. (N = 3–5, depicted as mean ± SD, independent two-tailed t-test). h. Pharmacological modulation of invasion with small GTPase inhibitors in UB068_CHRFAM7A isogenic monocytes. Monocytes were plated on Matrigel coated transwell inserts with or without CDC42 (ML141), RhoA (Rhosin) and Rac1 (EHT 1862) inhibitors. (N = 3–5, depicted as mean ± SD, independent two-tailed t-test). i. Representative images of UB068 and UB068_CHRFAM7A isogenic monocytes plated on fluorescent collagen for 16 h. Black pixels depict degradation. j. MMP2 gene expression levels of UB068 and UB068_CHRFAM7A monocytes at baseline and upon exposure to fibronectin coated hydrogel for 1 h (N = 3–5, depicted as mean ± SD, independent two-tailed t-test). k. Representative images of UB068 and UB068_CHRFAM7A isogenic macrophages incubated for 4 h with 1 μm polystyrene yellow green FluoSpheres. l. Quantification of time-dependent phagocytosis of 1 μm polystyrene yellow green FluoSpheres in UB068 and UB068_CHRFAM7A isogenic macrophages depicted as Box plots (N = 3, 30–50 cells/condition per experiment; Mann–Whitney U-test with Bonferroni multiple testing correction).