Protein kinase C-delta (PKC\u03b4), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice

S P Parihar,M Ozturk,T J Scriba,M J Marakalala,R Hurdayal,A Penn-Nicholson,D T Loots,R Guler,D Beukes Maasdorp,F Darboe,W A Hanekom,D E Zak,M Van Reenen,M Leitges,F Brombacher

doi:10.1038/mi.2017.68

Abstract

We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ−/− mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ−/− mice. Mechanistically, increased bacterial growth in macrophages from PKCδ−/− mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.

Highlights

Protein kinase C-d (PKCd) plays a multitude of physiological roles through its ability to phosphorylate multiple target proteins involved in various cellular processes such as signal transduction,[1] apoptosis,[2] proliferation and survival,[3] transcription,[4] hormonal regulation,[5] and immune responses.[6,7] PKCd À / À mice were originally characterized by two independent research groups who highlighted the role of this kinase in B-cell anergy[8] and B cell-mediated autoimmunity.[9]
PKCd mRNA expression was significantly higher in whole blood from 8 participants with pulmonary TB disease when compared with 16 QFT À healthy controls (QFT À ) but not with latent Mycobacterium tuberculosis (Mtb) infection (QFT þ ) (Figure 1b)
Using our publically available data set from CAGE sequencing of macrophages infected with a hypervirulent Mtb strain (HN878),[26] we reported that PKCd in the murine bone marrow-derived macrophages (BMDMs) was upregulated, whereas other PKC isoforms remained largely unaffected (Figure 6a), PKCd expression was validated by quantitative real time– PCR (qRT-PCR) in murine macrophages (Supplementary Figure S4a)

Summary

Introduction

Protein kinase C-d (PKCd) plays a multitude of physiological roles through its ability to phosphorylate multiple target proteins involved in various cellular processes such as signal transduction,[1] apoptosis,[2] proliferation and survival,[3] transcription,[4] hormonal regulation,[5] and immune responses.[6,7] PKCd À / À mice were originally characterized by two independent research groups who highlighted the role of this kinase in B-cell anergy[8] and B cell-mediated autoimmunity.[9]. A frequently used chemical rottlerin, which is a nonspecific chemical inhibitor of PKCd to block enzymatic reactions and intracellular signaling in pancreatic acinar cells.[13] Rottlerin was shown to reduce viral burdens in human monocyte-derived macrophages during the early stages of HIV-1 infection.[14] Physiologically, silencing of PKCd with anti-PKCd small interfering RNA resulted in increased cholesterol efflux in hamster kidney cells and a murine macrophage cell line RAW264.7.15 PKC isoforms such as d and e are required for phagocytosis in RAW264.7 macrophages,[16] and PKCe regulates autocrine production of tumor necrosis factor (TNF) that in turn induces apoptosis in lipopolysaccharide-activated macrophages.[17]

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Results

Conclusion