A cytoprotective role for optineurin during mycobacterial infection of macrophages

Abstract

Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor Optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn^+/+ and Optn^-/- mice with Mycobacterium smegmatis, and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn^-/- mice-derived BMDMs showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the Optineurin-deficient macrophages. Optineurin-deficient BMDMs showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for Optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, Optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1β. These observations taken together suggest a novel regulatory role for Optineurin during mycobacterial infection. Its deficiency leads to an impairment in macrophage responses, directly impacting the pathophysiology of infection.

Keywords: Autophagy; Cell death; Cytoprotectivity; Macrophages; Mycobacteria; Optineurin.

Graphical abstract

Fig. 1

Intracellular viability of M. smegmatis in Optn^−/−macrophages. CFU counts of M. smegmatis upon infection of Optn^+/+ and Optn^−/− BMDMs at an MOI of 10:1 (A) and 50:1 (B). (C) Optn^+/+ and Optn^−/− BMDM cell counts post 50:1 MOI M. smegmatis infection (n = 4 independent experiments, minimum 300 cells per experiment per genotype assessed) Bars represent mean ± SD. **p ≤ 0.01, ***p ≤ 0.001.

Fig. 2

Microscopic observation of nuclear morphology changes in M. smegmatis infected BMDMs at 50:1 MOI: (A) Cellular and nuclear morphology of uninfected BMDMs from Optn^+/+ and Optn^−/− mice. Cellular and nuclear morphology of M. smegmatis infected BMDMs from Optn^+/+ and Optn^−/− mice, 0 h (B), 8 h (C), and 20 h (D) post infection. (E). Quantification of BMDMs based on nuclear morphology into normal, pyknotic and pale staining nucleus. (n = 4 independent experiments, minimum 300 cells per experiment per genotype assessed) Bars represent mean ± SD. ***p ≤ 0.001.

Fig. 3

Assessment of autophagy in M. smegmatis infected BMDMs from Optn^+/+and Optn^−/−mice: (A) Representative Western blot showing the levels of LC3, Cleaved Caspase 3, Optineurin, and β-Actin in M. smegmatis infected BMDMs isolated from Optn^+/+ and Optn^−/− mice. (B) Bar diagram showing quantified levels of LC3 II, at 20 h post-infection. (n = 4 independent experiments) Bars represent mean ± SD. ***p ≤ 0.001. ‘#’ indicates a non-specific band. Con. UI = Uninfected Control macrophages.

Fig. 4

Levels of Pro-IL-1β in BMDMs from Optn^+/+and Optn^−/−mice: (A) Levels of Pro IL-1β in BMDMs isolated from Optn^+/+ and Optn^−/− mice on treatment with 1 μg/ml LPS. B) Bar diagram showing quantified levels of Pro IL-1β at 24 h post LPS treatment (n = 3 independent experiments). Bars represent mean ± SD. **p ≤ 0.01. ‘#’ indicates a non-specific band.