Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis

Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86(-/-) mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis.CD80(-/-) mice had improved survival after CLP when compared to WT or CD86(-/-) mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80(-/-) mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-kappaB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model.In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80

CD80 preferentially control lethality and inflammation in CLP.

<p>Panel A-WT (n = 20) and CD80^−/− (N = 15) and CD86^−/− (N = 15) underwent CLP and were monitored for survival. P<0.04 (CD80^−/− vs. CD86^−/−), p<0.001 (CD80^−/− vs. WT) and p<0.01 (WT vs. CD86^−/−). Panel B–D. WT and CD80^−/− and CD86^−/− mice underwent CLP and were sacrificed at 18 hrs and plasma (Panel B), BALF (Panel C) and PL (Panel D) were collected for cytokine analysis via commercially available ELISA. N = 8–10 mice/group. + = <0.1, * = <0.05.</p

CD80 blockade preferentially improves survival after CLP.

<p>Panel A-WT C57BL/6 mice were administered either 250 µg anti-CD80, anti -CD86 or both 4 hrs prior to CLP and monitored for survival (n = 10/group). Data from anti -CD80/86 has been published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006600#pone.0006600-Nolan1" target="_blank">[18]</a>. P = 0.002 for anti-CD80 vs. control. Panel B-WT mice were administered control IgG or anti -CD80 or anti -CD80/86 (250 µg each) 2 hrs post-CLP and monitored for survival (n = 10/group). p = 0.002 for anti-CD80 vs. control.</p

PMN membranes disassociate IRAK-M from CD80.

<p>Macrophages were treated with PMN lipid rafts for various times Panel A. Co-IP demonstrates CD80 binds IRAK-M in resting PMA differentiated THP-1 macrophages, lane 1. One hour after lipid raft treatment IRAK-M disassociates from CD80, lane 2. CD86 binds little IRAK-M in resting macrophages, lane3. There is association of IRAK-M with CD86 one hour after lipid raft addition, lane4. Panel B. Confocal microscopy of resting monocyte derived macrophages at baseline (Row 1,4) or stimulated with PMN lipid rafts for 1 hr (Row 2,5) or 4 hrs (Row 3,6). IRAK-M (Red), CD80 (Left Panel-green) or CD86 (Right Panel-green) and co-localization (Yellow). Nucelar stain with DAPI (blue). Panel C. PMA differentiated THP-1 macrophages were stimulated with PMN lipids rafts and harvested at the designated time point. Whole cell extracts were IP for TRAF-6 and blotted for IRAK-M. All lanes (panel A and C) were normalized for protein prior to immunoprecipitation.</p

Clinical Characteristics of enrolled subjects. Absolute values are presented are Mean±SEM.

<p>Clinical Characteristics of enrolled subjects. Absolute values are presented are Mean±SEM.</p

CD28^−/− mice are protected from lethality of polymicrobial sepsis.

<p>Panel A-WT (n = 15) and CD28^−/− (N = 15) underwent CLP and were monitored for survival. Panel B. WT and CD28^−/− mice underwent CLP and were sacrificed at 18 hrs and plasma collected for cytokine analysis via commercially available ELISA. N = 5/group.</p

Differential Regulation of CD80 and CD86 in human sepsis.

<p>Patients with sepsis (n = 31) had blood drawn within 24 hrs of ICU admission. Samples analyzed by flow cytometry and plasma underwent cytokine analysis. Panel A-CD14⁺ monocytes expression of CD80 in subjects with (n = 20) or without (N = 10) septic shock. Panel B- Correlation between CD14⁺ monocytes expression of CD80 and SOFA Score. Panel C- CD14⁺ monocytes expression of CD86 in survivors (N = 24) and non-survivors (N = 6). Panel D- Correlation between CD14⁺ monocytes expression of CD86 and ICU Free days. Panel E-CD14⁺ monocytes expression of CD86 in subjects with (n = 16) or without (N = 14) bacteremia on presentation. Panels F and G— Correlation between CD14⁺ monocytes expression of CD86 and IL-6 (Panel F) and IL-10 (Panel G).</p