Divergent host innate immune response to the smooth-to-rough M. abscessus adaptation to chronic infection

Abstract

Mycobacterium abscessus is a nontuberculous mycobacterium emerging as a significant pathogen for individuals with chronic lung disease, including cystic fibrosis and chronic obstructive pulmonary disease. Current therapeutics have poor efficacy. New strategies of bacterial control based on host defenses are appealing, but anti-mycobacterial immune mechanisms are poorly understood and are complicated by the appearance of smooth and rough morphotypes with distinct host responses. We explored the role of the complement system in the clearance of M. abscessus morphotypes by neutrophils, an abundant cell in these infections. M. abscessus opsonized with plasma from healthy individuals promoted greater killing by neutrophils compared to opsonization in heat-inactivated plasma. Rough clinical isolates were more resistant to complement but were still efficiently killed. Complement C3 associated strongly with the smooth morphotype while mannose-binding lectin 2 was associated with the rough morphotype. M. abscessus killing was dependent on C3, but not on C1q or Factor B; furthermore, competition of mannose-binding lectin 2 binding with mannan or N-acetyl-glucosamine during opsonization did not inhibit killing. These data suggest that M. abscessus does not canonically activate complement through the classical, alternative, or lectin pathways. Complement-mediated killing was dependent on IgG and IgM for smooth and on IgG for rough M. abscessus. Both morphotypes were recognized by Complement Receptor 3 (CD11b), but not CR1 (CD35), and in a carbohydrate- and calcium-dependent manner. These data suggest the smooth-to-rough adaptation changes complement recognition of M. abscessus and that complement is an important factor for M. abscessus infection.

Fig 1.. Whole plasma enhances 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; n=37; †, P = 1.2 × 10⁻¹¹, 8, 1.8×10⁻⁵), or D, incubated with BSA (n=4), and killing determined after 1 hr incubation with neutrophils. E, smooth, and F, rough Mab opsonized with the indicated percentage of WP or HIP and neutrophil killing determined after 1 hr incubation; n=5–6; *, P<0.05; **, P<0.01.

Fig 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 C3, C1q, and MBL2 were detected. A sample of WP was run as a positive control; * indicates C3 fragments. 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.

Fig 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 opsonized with WP were washed, proteins separated by SDS-PAGE, and deposited C3, 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 were opsonized with WP (blue) or HIP (orange) were added to human neutrophils at an MOI of 1 for 1 hr and killing determined; n = 4–11 per isolate.

Fig 4.. Opsonized killing of Mab requires C3, but is independent of CP and AP.

A, smooth Mab 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 hr and killing determined; n = 6–8. *, P<0.05; **P<0.01. C, smooth and rough Mab were opsonized with WP and the supernatants were retained. The washed cellular pellet and supernatant proteins were separated by SDS-PAGE, and deposited C3 was detected. A 1:20 dilution of WP was run as a positive control; representative of 3 independent experiments. 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 hr incubation; n = 6. F, smooth and rough Mab were opsonized in WP or WP + EDTA, washed, and deposited C3, C1q, IgG, and IgA were detected; n = 3. *, P<0.05; †, P<0.001.

Fig 5.. Opsonized killing of Mab is not inhibited by MBL2 ligands.

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 hr and killing determined; n= 5. B, Mab opsonized as in A were washed, proteins separated by SDS-PAGE, and deposited C3, MBL2, IgG, and IgM were detected. A 1:20 dilution of WP was run as a positive control.

Fig 6.. Immunoglobulins are required for 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 hr and killing determined. C, smooth and D, 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 hr and killing determined; n= 5–10 independent experiments. E, Mab opsonized as in Fig 2 were washed, proteins separated by SDS-PAGE, and deposited C3 and IgG were detected. A 1:20 dilution of WP or IgG-depleted WP (plasma) were run as positive controls; * indicates C3 fragments.; n= 5. *, P<0.05.

Fig 7.. CR3/CD11b and cations play independent roles in 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 hr and killing determined; n= 4–6. C, smooth and D, rough Mab opsonized with WP or HIP (solid) were added to human neutrophils pre-incubated with anti-CD35 (CR1) or anti-CD11b (CR3), as indicated, and killing determined; n= 5–6. *, P<0.05; **P<0.01.

Fig 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 hr 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 hr and killing determined; n=6 *, P<0.05; **P<0.01.