Fig 2: TRPC1 Mediates IFNγ-Induced Ca2+ Influx in Peritoneal Macrophages In Vivo(A) Schematic showing calcium imaging, electrophysiological recordings, and biochemical analysis performed on peritoneal macrophages (peritoneal macrophages) from IFNγ i.p. injected WT and TRPC1−/− mice or mice injected with TRPC1 siRNA or ORAI1 siRNA to transiently knock down these proteins in vivo before the animals received IFNγ.(B) Ca2+ entry triggered by Tg in peritoneal macrophages from WT or TRPC1−/− mice that received IFNγ i.p. (M1). Analog plots of the fluorescence ratio (340/380 nm) from an average of 40–50 cells are shown. The bar graph (B′) indicates means ± SEM of the Ca2+ release (left peak in B) and store-operated Ca2+ entry (SOCE) (right peak in B).(C) Ca2+ entry triggered by Tg in IFNγ-exposed peritoneal macrophages transiently deficient in TRPC1 (M1-siT1) or ORAI1 (M1-siO1), or control cells (M1-siC) obtained from mice that received non-targeting siRNA. Analog plots of the fluorescence ratio (340/380 nm) from an average of 40–50 cells are shown. The bar graph (C′) indicates means ± SEM of the Ca2+ release.(D) I-V curves in peritoneal macrophages from WT or TRPC1−/− mice that received IFNγ (M1) or vehicle (M0) i.p. Average of 8–10 recordings for current intensity at −80 mV is presented in the bar graph (D′).(E) IFNγ-exposed peritoneal macrophages transiently deficient in TRPC1 (M1-siT1) or ORAI1 (M1-siO1), or control cells (M1-siC) were subjected to whole-cell patch-clamp recordings. Average of 8–10 recordings used for I-V relationships are shown in bar graph (E′).(F) TRPC1-STIM1 and ORAI1-STIM1 complex formation in peritoneal macrophages from C57BL/6 mice i.p. injected with PBS (M0-PBS), thioglycolate (M0-Thio) and PBS, or thioglycolate and IFNγ (M1). Immunoprecipitation was performed on 250 μg of protein extracts from approx. 5 × 106 cells using anti-STIM1 antibodies (Cell Signaling, 4916S) as seen after subjecting the immunoprecipitates to SDS-PAGE followed by immunoblot detection with anti-TRPC1 (Abcam, ab192031) and anti-ORAI1(Alamone Lab, ACC-060). Anti-STIM1 (Cell Signaling, 4916S) was used for loading control. Bar graph (F′) represents the average pixel intensity of the respective protein bands from three independent experiments.(G) TRPC1 and ORAI1 protein expression in peritoneal macrophages from C57BL/6 mice i.p. injected with PBS (M0-PBS), thioglycolate and PBS (M0-Thio), or thioglycolate and IFNγ (M1) as seen subjecting 30 μg protein to SDS-PAGE and using anti-TRPC1 (Abcam, ab192031), anti-ORAI1 (Alamone Lab, ACC-060), or anti-GAPDH for western blot. Bar graph (G′) represents the average pixel intensity of the respective protein bands from three independent experiments.*p ≤ 0.05, ***p ≤ 0.001 (Student's t test).See also Figure S3.

Fig 3: TRPC1 Mediates Ca2+ Influx in Macrophage during Preclinical Peritonitis due to Klebsiella pneumoniae (KPn) Infection and in Human Patients with SIRS(A) Calcium imaging and electrophysiological recordings were performed on peritoneal macrophage from WT and TRPC1-/- mice i.p. infected with KPn for 24 hr.(A1) Peritoneal macrophages from WT and TRPC1-/- KPn-infected mice loaded with Fura-2AM and Tg added in Ca2+-free medium to measure the internal Ca2+ release (first peak) before addition of external Ca2+ as indicated to measure Ca2+ entry/influx through the plasma membrane (second peak). Analog plot of the fluorescence ratio (340/380 nm) from an average of 40–50 cells is shown. The bar graph (A2) indicates average ratio ± SEM of the Ca2+ release (first peak) and store-operated Ca2+ entry (SOCE) (second peak). (A3) Representative Ca2+ currents at -80 mV from a 0 mV holding potential of peritoneal macrophages from WT and TRPC1-/-KPn-infected mice by whole-cell patch-clamp analysis with 1 µM Tg in the pipette solution. (A4) I-V curves showing the TRPC1 channel-associated signature Isoc in peritoneal macrophages from KPn-infected WT, but not in TRPC1-/- mice. The signature current for ORAI1 channels (Icrac) was present in peritoneal macrophages from mock control WT and KPn-infected TRPC1-/- mice. Average current density recordings from 8 to 10 cells at -80 mV and corresponding statistics are shown in the bar graph (A5). (A6) From the KPn-infected WT and TRPC1-/- mice, peritoneal lavage, and liver and blood samples were collected. Liver was homogenized. Blood or liver homogenate or peritoneal lavage samples were serially diluted and plated on LB plates. Bacterial burden was enumerated after incubating the plates overnight at 37°C. Results shown here are mean ± SE from three experimental animals (n = 3). *p < 0.05**, p = 0.01, ***p = 0.001 (Student's t test).(B) M1 inflammatory activation phenotype in circulating monocytes/macrophage in humans with SIRS correlated with TRPC1 expression. Blood samples were collected from patients with SIRS every 24 hr for up to 10 days or until discharged from the ICU. PBMCs from patient and healthy donors (HC) were isolated and circulating monocytes/macrophages purified by positive magnetic selection. (B1) Western blot analysis using anti-TRPC1 and anti-ORAI1 was performed on cell lysates. GAPDH was used as loading control. The bar graph (B2) depicts averages ±SEM of pixel intensity of the TRPC1 and ORAI1 protein bands. Representative of n = 4 healthy donors and patients with SIRS. (B3) The expressions of M1-associated inflammatory mediators, CXCL9 and CXCL10, and M2 anti-inflammatory mediator, CCL22, were measured by qRT-PCR. *p < 0.05, **p = 0.01 (Student's t test).

Fig 4: Peritoneal Macrophage from TRPC1-/- Mice Exhibit Reduced M1 Inflammatory Responses during Experimental Peritonitis due to Klebsiella pneumoniae Infection(A) Peritoneal macrophages from mock control (M) and KPn-infected WT and TRPC1-/- mice were harvested. Western blot analysis using anti-pSTAT1, pp65, p65, STAT1, and GAPDH was performed on equal amount of the respective cell lysates. GAPDH, p65, and STAT1 were used as loading control. The bar graph (A') depicts averages ± SEM of pixel intensity of the pSTAT1 and pp65 protein bands.(B) Peritoneal macrophages from mock control (M) and KPn-infected WT and TRPC1-/- mice were harvested, and the relative mRNA expression of M1 inflammatory mediators, NOS2, TNF-?, IL-6, IL-23, CXCL9, and CXCL10, and M2 anti-inflammatory mediators, CCL22 and ARG-1, was measured by qRT-PCR.**p = 0.01, ***p = 0.001 (Student's t test).

Fig 5: Effect of TRPC1 Deficiency on the Ability of IFNγ to Induce M1 Macrophages In VitroTo analyze the effect of TRPC1 deficiency on M1 macrophage functions, BM macrophages from WT and TRPC1−/− mice were generated in vitro. In addition, BM macrophages from C57BL/6 mice were transfected with non-targeting siRNA or TRPC1 siRNA to transiently knock down TRPC1. Cells were cultured in the presence or absence of IFNγ, and the level of M1-associated signature immune mediators and transcriptions factors were measured by western blot, RT-PCR, and colorimetric assay.(A) BM macrophages transfected with non-targeting siRNA (siC), or TRPC1 siRNA (siT1) were pulsed with medium alone (M0-siC, M0-siT1) or IFNγ (M1-siC, M1-siT1). Immunoblot analysis were performed using anti-pSTAT1, anti-pNFκB p65 (pp65), STAT1, and p65. The average pixel intensity of the pSTAT1 and pp65 protein bands from three independent experiments is shown in A′.(B) NO was assessed by colorimetric assay in supernatants collected at 24 hr from SiC and siT1 cells treated as described in (A).(C) The relative mRNA expression of M1 inflammatory mediators, NOS2, IL-23, CXCL9, and CXCL10 in BM macrophages transfected with control siRNA (siC) or TRPC1 siRNA (siT1) and pulsed for 24 hr with IFNγ (M1) versus medium only (M0).(D) The relative mRNA levels of M1 inflammatory mediators, NOS2, TNF-ᾳ, IL-6, IL-23, CXCL9, and CXCL10, and M2 anti-inflammatory mediators, CCL22 and arginase-1 (ARG-1), were analyzed in BM macrophages from WT and TRPC1−/− mice and pulsed for 24 hr with IFNγ (M1) versus medium only (M0).(E) BM macrophages transfected with control siRNA or TRPC1 siRNA and pulsed for 24 hr with medium only (M0-siC, M0-siT1) or IFNγ (M1-siC, M1-siT1). The surface expression of costimulatory molecule CD86 was measured by flow cytometry. Plots in (E′) depict the mean ± SEM of M0-siC, M0-siT1, M1-siC, and M1-siT1 cells expressing MHC-II, CD80, or CD86 as measured by flow cytometry (density plots shown in E and Figure S4B).*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (Student's t test).See also Figures S4–S7.