Oxidation-Specific Epitopes (OSEs) Dominate the B Cell Response in Murine Polymicrobial Sepsis

Abstract

In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis.

Keywords: B cell response; CASP; oxidation-specific epitopes; polymicrobial sepsis; polyreactive antibodies.

Figure 1

Binding of sera IgM/IgG to bacteria and LPS. The binding of murine serum antibodies, IgM (A-D) or IgG (E-H), to indicated bacteria; E. coli (A,C), E. faecalis (B,F), S. aureus (C,G), and LPS (D,H) was tested by ELISA. Fourteen days after sepsis induction, blood was collected, and sera were diluted 1:100 in blocking buffer. Sera from non-septic animals were used as controls. Control mice were untreated (•), whereas the treated mice were anesthetized only (▴), or additionally received a laparotomy (), or sham surgery (▵). Mice that underwent CASP surgery are indicated by a black diamond (♦). Mean values of OD 450 nm are shown. Each symbol represents one animal (N = 5–18). Statistical analysis was done by One-way ANOVA with the Bonferroni post test for selected pairs. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Serum antibodies directed against self-antigens 14 days after sepsis induction. Sepsis was induced in C57BL/6 mice by 18G CASP. Control mice remained untreated. Fourteen days later blood was taken, and sera diluted 1:100 in 20% FCS/PBS were incubated on HEp-2-ANA slides. Bound antibodies were detected by FITC-labeled anti-mouse IgM or IgG antibodies. Out of the tested serum antibodies, 12/13 IgM and 12/13 IgG antibodies were autoreactive. Shown are representative pictures of septic and control mice. N = 8 per group. Serum IgM and IgG binding patterns varied greatly. In most cases more than one cellular structure is stained, albeit at variable intensities.

Figure 3

Serum IgM and IgG binding to model oxidation-specific epitopes. C57BL/6 mice were subjected to CASP surgery, while control animals remained untreated. Ten and 14 days after sepsis induction, blood was drawn to determine serum IgM and IgG binding to malondialdehyde-modified LDL [MDA-LDL; (A)] and phosphocholine-conjugated BSA [PC-BSA; (B)], using chemiluminescent ELISA. Serum dilutions used are indicated in brackets. Statistical analysis was performed using the unpaired t-test. N = 3–9 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Monoclonal antibodies derived from splenic B cells of septic mice bind several cellular self-antigens. Ten or 14 days after CASP induction, splenocytes were isolated and fused with myeloma cells to obtain hybridomas. Supernatants of monoclonal IgM- or IgG-producing hybridomas were incubated on HEp-2-ANA slides to detect auto-reactivity. Bound IgG or IgM was detected by FITC-labeled anti-mouse IgG or IgM antibodies. Three examples, respectively, of autoreactive monoclonal IgM- and IgG-antibodies are shown. The binding patterns of the various antibodies showed obvious differences, where in most cases more than one cellular structure was stained, albeit at varying intensities (indicated by arrows). For quantified data, regarding the numbers/percent of IgM or IgG hybridoma supernatants from septic mice directed against self-antigens, please refer to Table 2.

Figure 5

Number of mutations in Vh-genes of monoclonal IgG. CASP surgery was performed on C57BL/6 mice. Hybridomas were generated by fusing splenocytes from 10- days-septic mice. The resultant monoclonal IgG hybridomas were sequenced for Vh-genes. Mutations of monoclonal IgG that showed binding to OSEs (OSE pos.) were compared to IgG that showed no binding (OSE neg.). Statistical analysis was performed using the unpaired t-test. N = 5–14 per group. nt, nucleotides; ns, not significant.