Figure 6.

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 I_soc in peritoneal macrophages from KPn-infected WT, but not in TRPC1^−/− mice. The signature current for ORAI1 channels (I_crac) 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).