Sepsis-induced potentiation of peritoneal macrophage migration is mitigated by programmed cell death receptor-1 gene deficiency

Abstract

The effect of programmed cell death receptor-1 (PD-1) on phagocyte function has not been extensively described. Here we report that experimental mouse sepsis, cecal ligation and puncture (CLP), induced a marked increase in peritoneal macrophage random migration, motility and cell spread, but these changes were lost in the absence of PD-1. Alternatively, phagocytic activity was inversely affected. In vitro cell culture imaging studies, with the macrophage cell line J774, documented that blocking PD-1 with antibody led to aggregation of the cytoskeletal proteins α-actinin and F-actin. Further experiments looking at ex vivo peritoneal macrophages from mice illustrated that a similar pattern of α-actinin and F-actin was evident on cells from wild-type CLP mice but not PD-1-/- CLP mouse cells. We also observed that fMLP-induced migration by J774 cells was markedly attenuated using PD-1 blocking antibodies, a nonselective phosphatase inhibitor and a selective Ras-related protein 1 inhibitor. Finally, peritoneal macrophages derived from CLP as opposed to Sham mice demonstrated aspects of both cell surface co-localization with CD11b and internalization of PD-1 within vacuoles independent of CD11b staining. Together, we believe the data support a role for PD-1 in mediating aspects of innate macrophage immune dysfunction during sepsis, heretofore unappreciated.

Fig. 1

CLP-induced rise in peritoneal macrophage migratory capacity is reduced by PD-1 gene deficiency. a Representative tracing of mouse peritoneal macrophage migration over time of cells derived from septic PD-1−/− mice as compared to cells from septic WT mice (48 h post-CLP). b At 24 h, CLP mice macrophages migrate further, but there is no difference in the absence of PD-1. However, at 48 h, CLP PD-1−/− mice had greatly reduced migratory distance when compared to CLP WT mouse cells, but again with no differences in the Sham groups. c Similar results were seen in motility. * p < 0.05 versus PD-1−/− CLP (n = 5-6 per group), determined by ANOVA followed by Tukey's test.

Fig. 2

CLP-induced increase in the number of spread macrophage per field is reduced by PD-1 gene deficiency. a Representative images of macrophage monolayers derived from WT and PD-1−/− CLP illustrating the reduced number of spread cells typically seen on ICAM-1-coated plates (×20). b Summary data number of spread cells per field at 0 and 15 min (mean ± SEM for 3 repeat experiments), which illustrates that the macrophages derived from CLP WT mice exhibited a marked increase in the number of spread cells per field when compared to cells from PD-1−/− CLP mice. * p < 0.05 WT versus PD-1−/−; # p < 0.05 15 min group versus 0 time; † p < 0.05 Sham versus CLP; determined by the Mann-Whitney rank-sum test.

Fig. 3

CLP-induced suppression of bacterial phagocytosis is reversed by PD-1 gene deficiency. a Typical FACS histograms of peritoneal macrophages that had been isolated by adherence from WT Sham, WT CLP, PD-1−/− Sham or PD-1−/− CLP mice and then co-cultured with pHrodo™-conjugated E. coli BioParticles® for 1 h. Cells were detached and assessed for their extent of E. coli BioParticles® fluorescence intensity (mean fluorescence intensity is provide in parentheses) by flow cytometry. A region, M1, in which a cell was considered positive for containing fluorescent E. coli BioParticles®, was established by comparison with a negative control that was run at 4°C as opposed to the 37°C used in all other cases. b Summary data for repeated assessments of macrophage preparations from 6-10 independent animals for a given treatment group. Data is presented as mean ± SEM. Asterisk (*) indicates the presence of a significant difference (p < 0.05) between treatment groups as determined by the Mann-Whitney rank-sum test.

Fig. 4

J774 cells exhibit altered cytoskeletal immunefluorescent staining pattern for either anti-α-actinin or anti-F-actin when incubated with anti-PD-1 blocking antibody but not control IgG. a Staining J774 cells with anti-PD-1 show that ∼97% of cells are PD-1+, as determined by flow cytometry and depicted in the representative histogram. b, c Differences typically observed in the α-actinin-stained cells [in green for FITC- vs. DAPI-stained nuclei (blue); magnification ×40] treated with a non-specific IgG (control antibody; b) versus those treated with anti-PD-1 (c). Arrows point to area aggregations that contribute to a punctate pattern of their staining for α-actinin. d, e Illustration of the staining pattern typically observed in the F-actin-stained cells [in green for FITC- vs. DAPI-stained nuclei (blue); magnification ×40] treated with a non-specific-IgG (control antibody; d) versus those treated with anti-PD-1 (e).

Fig. 5

Experimental sepsis induces alterations in peritoneal macrophage cytoskeletal staining that are attenuated in cells derived from mice lacking PD-1. Peritoneal macrophages derived from WT CLP mice (after 24 h), but not cells from PD-1−/− CLP mice, exhibit a more irregular and spread cell morphology and a punctate pattern of fluorescent α-actinin (arrows; a-d) or F-actin (e-h) staining (in green for FITC) that is not seen in either of the treated Sham group's cells (DAPI nuclear counter stain in blue).

Fig. 6

Sepsis induces increased expression of PD-1 and its ligand on F4/80+ cells. a Experimental sepsis produced by CLP induces upregulation of the percentage of WT mouse peritoneal macrophages (gated as F4/80+) expressing PD-1. b While mice lacking the PD-1 gene (PD-1−/−) product exhibited no detectable PD-1 on their peritoneal macrophages (left), they still upregulated the percentage of macrophages that were PD-L1+ to the same extent and fashion as WT CLP animal cells (right). Summary data is presented as mean ± SEM derived from n = 6-8 independent mouse macrophage preparations assessed per treatment group. Asterisk (*) indicates the presence of a significant difference when p < 0.05 between CLP and Sham treatment for the equivalent genetic mouse background animals as determined by the Mann-Whitney rank-sum test. ND = Not detected.

Fig. 7

Treatment of J774 cells with either antibody to inhibit PD-1 ligation, or pharmacological inhibitors to its downstream signaling, inhibit cell migratory capacity. Determination of the effect of anti-PD-1 blocking antibody (10 or 20 μg/ml), a Rap1 inhibitor (Ggti at 50 μM) or the phosphatase inhibitor (orthovanadate at 10 μM) on fMLP (1 μM) induced J774 cell TransWell migration (see Methods for details). Summary data is presented as mean ± SEM from 3 repeat experiments. * p < 0.05 versus no treatments and no fMLP stimulation control group (far left open bar group); # p < 0.05 versus fMLP stimulation alone control group (2nd solid black bar on left side); as determined by ANOVA followed by Tukey's test.

Fig. 8

PD-1 co-localizes with CD11b on septic mouse peritoneal macrophages. a Representative three-color immunofluorescence staining of adherent peritoneal macrophages derived from Sham or CLP WT mice at 24 h post-CLP for the nucleus (DAPI), CD11b (anti-CD11b) and PD-1 (anti-PD-1) co-localization. b Results of a typical Western immunoblot analysis for PD-1 expression on immunoprecipitated protein (IP) samples obtained from the cell lysates of peritoneal macrophages harvested from Sham versus CLP mice using anti-mouse CD11b antibody or IgG control. c Summary/repeated integrated densitometric values (IDV) of PD-1 expression for CD11b IP samples are provided in the histogram. Data is presented as mean ± SEM derived from n = 4-8 independent mouse macrophage preparations assessed per treatment group. Asterisk (*) indicates the presence of a significant difference (p < 0.05) between CLP and Sham treatment animals as determined by the Mann-Whitney rank-sum test.

Fig. 9

A proposed model of how we believe the increase in septic WT mouse peritoneal macrophage expression of PD-1 (and the comparative effect of its absence in PD-1−/− mice) may affect the function of these leukocytes.