Methods: We magnetically sorted CD14+monocytes from whole blood for functional assays including calcium flux to assess receptor signaling, flow cytometry to assess receptor expression and cell apoptosis/death, alamarBlue-based fungal killing, and cytokine production post-fungal stimulation measured by Luminex array. Data were analyzed using a Mann-Whitney or t-test where appropriate.
Results: We screened 108 healthy Caucasian donors from the NIH Blood Bank and identified 67 wild-type (WT), 37 CX3CR1-M280 heterozygous, and 4 CX3CR1-M280 homozygous donors. We found that CX3CR1-M280 homozygotes exhibit a 50% decrease in FKN-induced receptor signaling (p=0.0002), which was not due to decreased CX3CR1 surface expression. Consistent with our prior mouse findings that Cx3cr1 mediates anti-apoptotic effects in macrophages, we found that FKN was able to rescue serum starvation-induced monocyte death in WT donors (p=0.0031) but not in CX3CR1-M280 homozygotes (p=ns). This survival defect in CX3CR1-M280 homozygote monocytes may account for the observed steady state monocytopenia seen in these donors (p=0.0178). Instead, CX3CR1-M280 did not impair monocyte fungal killing or production of pro-inflammatory cytokines in response to heat-killed Candida.
Conclusion: Together, these data show that the CX3CR1-M280 allele impairs FKN-induced CX3CR1 signaling in human monocytes and results in defective monocyte survival and monocytopenia.
M. Lionakis, None