Porcine CXCR1/2 antagonist CXCL8(3-72)G31P inhibits lung inflammation in LPS-challenged mice.

Swine pneumonia is a great threat for pig industry around the world, which is usually accompanied with neutrophils infiltration in the airway. Although interleukin-8 (CXCL8) and its receptors, CXC chemokine receptor 1 and 2 (CXCR1/2) in human have been well documented, the expression and function of CXCR1/2 is still unknown in swine. To explore the feasibility to develop new veterinary anti-inflammatory drugs targeting porcine CXCR1/2, we detected CXCR1/2 expression in swine pneumonia through Real-Time PCR and immunohistochemistry for the first time. Two porcine CXCR1/2 antagonists, CXCL8(3-72)N11R/G31P (pN11R) and CXCL8(3-72)G31P (pG31P) were prepared and their anti-inflammatory effects were evaluated using cell chemotaxis assays and animal experiments. Our data showed that CXCR1/2 expression, which was closely related to neutrophil infiltration in the lung, was significantly up-regulated in swine pneumonia. The pN11R and pG31P could effectively inhibit the directional migration of neutrophils in vitro. In vivo data also indicated that both pN11R and pG31P significantly relieved LPS-induced pneumonia in mice through decreasing the expression of TNF-α, CXCL8, and IL-1ß, and inhibiting neutrophil influx into the lung. pG31P was more efficient. Our study suggested that it is possible to develop new veterinary anti-inflammatory drugs targeting porcine CXCR1/2, and pG31P is a promising candidate.

A Neutrophil Phenotype Model for Extracorporeal Treatment of Sepsis.

Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage in sepsis. Interleukin-8 (IL-8), a key modulator of neutrophil function, signals through neutrophil specific surface receptors CXCR-1 and CXCR-2. In this study a mechanistic computational model was used to evaluate and deploy an extracorporeal sepsis treatment which modulates CXCR-1/2 levels. First, a simplified mechanistic computational model of IL-8 mediated activation of CXCR-1/2 receptors was developed, containing 16 ODEs and 43 parameters. Receptor level dynamics and systemic parameters were coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils which reduce pathogen load, and/or primed neutrophils which cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a two-hour infusion of E coli and followed for a maximum of 28 days. Ensembles of parameters were generated using a Bayesian parallel tempering approach to produce model fits that could recreate experimental outcomes. Stepwise logistic regression identified seven model parameters as key determinants of mortality. Sensitivity analysis showed that parameters controlling the level of killer cell neutrophils affected the overall systemic damage of individuals. To evaluate rescue strategies and provide probabilistic predictions of their impact on mortality, time of onset, duration, and capture efficacy of an extracorporeal device that modulated neutrophil phenotype were explored. Our findings suggest that interventions aiming to modulate phenotypic composition are time sensitive. When introduced between 3-6 hours of infection for a 72 hour duration, the survivor population increased from 31% to 40-80%. Treatment efficacy quickly diminishes if not introduced within 15 hours of infection. Significant harm is possible with treatment durations ranging from 5-24 hours, which may reduce survival to 13%. In severe sepsis, an extracorporeal treatment which modulates CXCR-1/2 levels has therapeutic potential, but also potential for harm. Further development of the computational model will help guide optimal device development and determine which patient populations should be targeted by treatment.

Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination.

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.

Cloning and characterization of guinea pig interleukin-8 receptor.

CXC-chemokine receptors 1 and 2 and their ligands (CXCL1, 2, 3, 5, 6, 7, and 8) induce the selective recruitment of neutrophils during inflammation. Such receptors have not been characterized yet in guinea pig, an animal inflammation model of interest. We report the identification, cloning, and characterization of a CXCL8 receptor in guinea pig. Human CXCL8 produced in vivo neutrophilia, chemotaxis and intracellular calcium release of guinea pig neutrophils. The expression of this receptor at their neutrophil surface was investigated. The cDNA encoding a functional CXCL8 receptor was cloned from guinea pig neutrophils and sequenced. It was synthesized using RT-PCR, with oligonucleotide primers derived from well conserved regions of published CXCL8 receptors. This sequence presented an open reading frame coding for 352 amino acids and shares, at the amino acid level, 70 and 69% identity with human and rabbit CXCR2, respectively. The receptor was mainly expressed in neutrophils but it was also present in kidney, lung, spleen and, to a less extent, in heart. Cloned receptor transfected cells showed that this receptor displayed high affinity for human CXCL8, slightly lower than the affinity observed with guinea pig neutrophils. CXC chemokines from both rabbit and human were shown to induce inositol phosphate accumulation in these transfected cells. Receptor binding and activation characteristics together with sequence homology suggested that we identified a guinea pig equivalent of the human CXCR2 receptor.