Divergent host humoral innate immune response to the smooth-to-rough adaptation of Mycobacterium abscessus in chronic infection

Abstract

Mycobacterium abscessus is a nontuberculous mycobacterium emerging as a significant pathogen in individuals with chronic lung diseases, including cystic fibrosis and chronic obstructive pulmonary disease. Current therapeutics have poor efficacy. Strategies of bacterial control based on host defenses are appealing; however, antimycobacterial immunity remains poorly understood and is further complicated by the appearance of smooth and rough morphotypes, which elicit distinct host responses. We investigated the role of serum components in neutrophil-mediated clearance of M. abscessus morphotypes. M. abscessus opsonization with complement enhanced bacterial killing compared to complement-deficient opsonization. Killing of rough isolates was less reliant on complement. Complement C3 and mannose-binding lectin 2 (MBL2) were deposited on M. abscessus morphotypes in distinct patterns, with a greater association of MBL2 on rough M. abscessus. Killing was dependent on C3; however, depletion and competition experiments indicate that canonical complement activation pathways are not involved. Complement-mediated killing relied on natural IgG and IgM for smooth morphotypes and on IgG for rough morphotypes. Both morphotypes were recognized by complement receptor 3 in a carbohydrate- and calcium-dependent manner. These findings indicate a role for noncanonical C3 activation pathways for M. abscessus clearance by neutrophils and link smooth-to-rough adaptation to complement activation.

Keywords: adaptation; complement; cystic fibrosis; natural antibodies; neutrophils.

Figure 1

Whole plasma enhances the killing of Mab. (A) Smooth (Sm) Mab and (B) rough (R) Mab opsonized with whole plasma (WP; blue) or heat-inactivated plasma (HIP; orange) were added to human neutrophils at an MOI of 1 for the indicated times and killing determined; n = 3. (C) Smooth and rough Mab were opsonized in WP or HIP, or (D) incubated with BSA and killing determined after 1 h of incubation with neutrophils (n = 37 for Sm and R, and n = 4 for BSA). (E) Smooth and (F) rough Mab opsonized with the indicated percentage of WP or HIP and killing determined after 1 h of incubation with neutrophils (n = 5–6: ^* p < 0.05; **p < 0.01: ^* p < 0.05; ^† p < 0.001).

Figure 2

Whole plasma differently opsonizes Mab morphotypes. (A) Smooth (Sm) and rough (R) Mab opsonized with whole plasma were washed, proteins separated by SDS-PAGE, and deposited iC3b, C1q, and MBL2 were detected. A 1:20 dilution of WP was run as a positive control; Asterisk indicates iC3b reactivity at 70, 130, and 260 kDa. (B) Detection of IgG (G) and IgA (A). (C) Detection of IgM (M). The results are representative of over 10 separate experiments. Molecular weight markers are shown to the left of each blot.

Figure 3

Killing of smooth Mab clinical isolates is more dependent on complement than rough Mab isolates. (A) Smooth (S) and rough (R) Mab clinical isolates (numbered by isolate) opsonized with WP were washed, proteins separated by SDS-PAGE, and deposited iC3b, C1q, MBL2, IgG, and IgM were detected. A 1:20 dilution of WP was run as a positive control. S and R designate the morphotype. ^* p < 0.05; ^** p < 0.01. (B) Smooth and rough Mab clinical isolates opsonized with WP (blue) or HIP (orange) were added to human neutrophils at an MOI of 1 for 1 h and killing determined; n = 4–11 per isolate.

Figure 4

The opsonized killing of Mab requires C3 but is independent of CP and AP. (A) Smooth and (B) rough Mab opsonized with serum, sera depleted of C3, C1q, and Factor B, or HIP were added to human neutrophils at an MOI of 1 for 1 h and killing determined; n = 6–8. (C) Smooth and rough Mab were opsonized with WP and the supernatants retained. The washed cellular pellet and supernatant proteins were separated by SDS-PAGE, and deposited iCb3 was detected. A 1:20 dilution of WP was run as a positive control; n = 3–5. (D) Smooth and (E) rough Mab were opsonized with WP or HIP alone or in the presence of Mg²⁺/EDTA or EGTA before adding to neutrophils. Killing was determined after 1 h of incubation; n = 6. (F) Smooth and rough Mab were opsonized in WP or WP + EDTA, washed, and deposited iC3b, C1q, IgG, and IgA were detected; n = 3. ^* p < 0.05; ^† p < 0.001.

Figure 5

MBL2 ligands present during opsonization do not affect the killing of Mab. (A) Smooth and rough Mab opsonized with WP alone (solid) or in the presence of mannan (diagonal stripes; 1 mg/ml) or GlcNAc (horizontal stripes; 100 mM) were added to human neutrophils at an MOI of 1 for 1 h and killing determined; n = 5. (B) Mab opsonized as in (A) were washed, proteins separated by SDS-PAGE, and deposited iC3b, MBL2, IgG, and IgM were detected; n = 5. A 1:20 dilution of WP was run as a positive control. Asterisk indicates immunopositive bands.

Figure 6

Immunoglobulins are required for the killing of Mab opsonized in WP. (A) Smooth and (B) rough Mab opsonized with WP or HIP alone (solid) or in WP or HIP depleted of IgM with anti-IgM agarose (striped bars) were added to human neutrophils at an MOI of 1 for 1 h and killing determined. (C) Mab opsonized as for (A, B) were washed, proteins separated by SDS-PAGE, and deposited iC3b, MBL2, IgG, and IgM were detected. A 1:20 dilution of WP or IgM-depleted WP (plasma) was run as a positive control. The asterisk indicates C3 fragments. (D) Smooth and (E) rough Mab opsonized with WP or HIP alone (solid) or in WP or HIP depleted of IgG with Protein G Sepharose (striped bars) were added to human neutrophils at an MOI of 1 for 1 h and killing determined; n = 5–10 independent experiments. (F) Mab opsonized as in (D, E) were washed, proteins separated by SDS-PAGE, and deposited iC3b and IgG were detected. A 1:20 dilution of WP or IgG-depleted WP (plasma) was run as a positive control. The asterisk indicates C3 fragments. n = 5. ^* p < 0.05.

Figure 7

CR3/CD11b and cations play independent roles in the killing of opsonized Mab. (A) Smooth and (B) rough Mab opsonized with WP or HIP (solid) were added to human neutrophils in the presence of EDTA (striped bars) or EGTA (light shading) at an MOI of 1 for 1 h and killing determined; n = 4–6. (C) Smooth and (D) rough Mab opsonized with WP or HIP (solid) were added to human neutrophils preincubated with anti-CD35 (CR1) or anti-CD11b (CR3), as indicated, and killing determined; n = 5–6. ^* p < 0.05; ^** p < 0.01.

Figure 8

Inhibition of Mab killing by N-acetylated sugars. (A) Smooth and (B) rough Mab opsonized with WP or HIP alone (solid) or incubated with BSA were added to human neutrophils in the presence of GlcNAc (diagonal stripes; 100 mM) or GalNAc (horizontal stripes; 100 mM) for 1 h and killing determined; n = 5. (C) Smooth and (D) rough Mab opsonized with WP or HIP alone (solid) were added to human neutrophils in the presence of mannan (diagonal stripes; 1 mg/ml) for 1 h and killing determined; n = 6; ^* p < 0.05; ^** p < 0.01.