CD14 and complement crosstalk and largely mediate the transcriptional response to Escherichia coli in human whole blood as revealed by DNA microarray - GSE55537

Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation.

A microarray study was performed on whole blood from two healthy donors, a C5-deficient patient (C5D) (with and without C5-reconstitution) incubated for 120 min at 37oC with (i) PBS, (ii) E. coli or (iii) E. coli and inhibitors of complement (C3 or C5aR) and/or CD14 or inhibitor controls. The experiments were performed as biological replicates on two different days (healthy donor and C5D). Due to lack of a second C5-deficient patient, microarray analyses were prerformed twice (technical replicate) for C5D and day 1. Expression data in presence of PBS were used as reference for those in pressence of E. coli. Expression data in presence of E. coli were used as reference for those in pressence of E. coli and inhibitors. Expression data from 0 min incubation with PBS represent the initial state (T0). Whole blood was lysed in Applied Biosystems AB6100 total RNA chemistry lysis buffer and stored at -80oC prior to RNA extraction.

E. coli exposure (2021 ICD-10-CM code* = Not Applicable)

Robust Multichip Analysis by Partek® Genomics Suite software™ (transcripts with log2(signal value)<4 were excluded from the list)

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55537

sufficent_none_none (S_N_N)

sufficent_1 x E06/mL E. coli_none (S_1M_E_N)

sufficent_5 x E06/mL E. coli_none (S_5M_E_N)

sufficent_1 x E06/mL E. coli_Compstatin (S_1M_E_C)

sufficent_5 x E06/mL E. coli_Compstatin (S_5M_E_C)

sufficent_1 x E06/mL E. coli_anti-CD14 (S_1M_E_aC)

sufficent_5 x E06/mL E. coli_anti-CD14 (S_5M_E_aC)

sufficent_1 x E06/mL E. coli_anti-CD14 and Compstatin (S_1M_E_aC_C)

sufficent_5 x E06/mL E. coli_anti-CD14 and Compstatin (S_5M_E_aC_C)

sufficent_1 x E06/mL E. coli_C5a receptor antagonist (S_1M_E_C5a)

sufficent_5 x E06/mL E. coli_C5a receptor antagonist (S_5M_E_C5a)

sufficent_1 x E06/mL E. coli_inhibitor controls (S_1M_E_In)

sufficent_5 x E06/mL E. coli_inhibitor controls (S_5M_E_In)

deficient_none_none (D_N_N)

deficient_1 x E06/mL E. coli_none (D_1M_E_N)

deficient_5 x E06/mL E. coli_none (D_5M_E_N)

deficient_1 x E06/mL E. coli_Compstatin (D_1M_E_C)

deficient_5 x E06/mL E. coli_Compstatin (D_5M_E_C)

deficient_1 x E06/mL E. coli_anti-CD14 (D_1M_E_aC)

deficient_5 x E06/mL E. coli_anti-CD14 (D_5M_E_aC)

deficient_1 x E06/mL E. coli_anti-CD14 and Compstatin (D_1M_E_aC_C)

deficient_5 x E06/mL E. coli_anti-CD14 and Compstatin (D_5M_E_aC_C)

deficient_1 x E06/mL E. coli_C5a receptor antagonist (D_1M_E_C5a)

deficient_5 x E06/mL E. coli_C5a receptor antagonist (D_5M_E_C5a)

deficient_1 x E06/mL E. coli_inhibitor controls (D_1M_E_In)

deficient_5 x E06/mL E. coli_inhibitor controls (D_5M_E_In)

C5 reconstituted_none_none (R_N_N)

C5 reconstituted_1 x E06/mL E. coli_none (R_1M_E_N)

C5 reconstituted_5 x E06/mL E. coli_none (R_5M_E_N)

C5 reconstituted_1 x E06/mL E. coli_Compstatin (R_1M_E_C)

C5 reconstituted_5 x E06/mL E. coli_Compstatin (R_5M_E_C)

C5 reconstituted_1 x E06/mL E. coli_anti-CD14 (R_1M_E_aC)

C5 reconstituted_5 x E06/mL E. coli_anti-CD14 (R_5M_E_aC)

C5 reconstituted_1 x E06/mL E. coli_anti-CD14 and Compstatin (R_1M_E_aC_C)

C5 reconstituted_5 x E06/mL E. coli_anti-CD14 and Compstatin (R_5M_E_aC_C)

C5 reconstituted_1 x E06/mL E. coli_C5a receptor antagonist (R_1M_E_C5a)

C5 reconstituted_5 x E06/mL E. coli_C5a receptor antagonist (R_5M_E_C5a)

C5 reconstituted_1 x E06/mL E. coli_inhibitor controls (R_1M_E_In)

C5 reconstituted_5 x E06/mL E. coli_inhibitor controls (R_5M_E_In)