Protein Kinase C δ: a critical hub regulating macrophage immunomodulatory functions during Mycobacterium tuberculosis infection

Abstract

A host-modulating candidate gene involved in putative pathogen-killing pathways, with potential novel therapeutic intervention, Protein Kinase C - δ (PKCδ) has been recognized as a critical marker of inflammation with clinical and experimental evidence in recent years. Pulmonary microenvironment during Mtb infection is largely governed by lung resident macrophages, initiating innate and subsequent adaptive immune responses. We investigated the role of PKCδ in macrophages using a macrophage-specific PKCδ knockout mice model (LysM^crePKCδ^flox/flox). PKCδ deficiency in macrophages triggers an early lymphocytic immune response, increases neutrophil recruitment, and reduces inflammatory macrophages in the lungs, leading to higher Mtb burden and exacerbated pathology. Experimental and omics analysis further revealed that dysregulation of antimicrobial effector functions is detrimental to macrophage's ability to restrict bacterial growth in vitro. Importantly this defect was mitigated by exogenous GM-CSF supplementation and/or overexpressing PKCδ in macrophages. Thus, PKCδ plays a crucial role in immune modulation during Mtb infection with GM-CSF amongst several downstream pathways through which PKCδ exerts its regulatory effects.

Figure 1.. PKCδ deficiency in macrophages increased Mtb burden and worsened lung pathology in vivo.

[A] Schematic of LysM^crePKCδ^flox/flox mice generation and experimental outline of time course study. HN878 Mtb strain was challenged by intranasal route to LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 150 CFU/mouse. [B-C] Animals were euthanized to determine lung/spleen CFU burden at acute stage (4 wpi) and chronic stage (12 wpi) of Mtb infection. [D-E] Quantification of alveolar space represented with their respective (40X) scanned images (Scale bar = 1000 μm). [F-G] Quantification of iNOS+ area represented with their respective (40X) scanned images (Scale bar = 100 μm). [H-I] Determination of IL-2, IL-10 and GM-CSF levels by ELISA in the lung homogenates of LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 4 wpi and 12 wpi Mtb infection. [J-M] Flow cytometry analysis of myeloid subsets, CD4/CD8 effector memory (CD44+CD62L−), CD8 short lived effector cells (SLEC) (KLRG1+CD127−), and CD4/CD8 Exhaustion (PD1-KLRG1+) reported as frequency of live cells at 4 wpi of Mtb infection. All data shown mean+/− SD and is representative of two independent experiments with n=4–6 mice/group. Statistical analyses were performed using an unpaired student t-test. Asterisks are defining significance compared to the control group as: *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2.. Ablation of PKCδ in macrophages led to varying antimicrobial effector functions in vitro.

[A] Determination of mycobacterial burden in bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at indicated time points. [B] Determination of mycobacterial burden in PKCδ-overexpressed RAW264.7 murine macrophage cells at indicated time points. [C-D] Accumulation of cellular and mitochondrial ROS were determined at indicated time points in bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice during Mtb infection. [E] iNOS expression and subsequent [F] nitrite levels in bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at indicated time points. [G-H] Determination of Mean Fluorescence Intensity (MFI) of apoptotic marker Caspase-3 and macrophage activation markers (CD80, CD86, MHCII) by flow cytometry in bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 24 hours post Mtb infection. [I-K] Determination of pro-inflammatory cytokine (IL-1α, IL-1β, IL-6) levels in bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice in Mtb infection. [L-Q] mRNA expression of inflammasome pathway mediators (Caspase-1, Caspase-11, NLRP3, AIM2, IL-18 and IL-1β) in Mtb-infected bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice. [R-S] Total ATP production rate and metabolic dependence of bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at naive state and 24 hours post Mtb infection. [T-U] Determination of Mean Fluorescence Intensity (MFI) of macrophage phenotypic M1 and M2 marker by flow cytometry in bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 24 hours post Mtb infection. All data shown mean+/− SD and is representative of two independent experiments performed in quadruplicate. Statistical analyses were performed using an unpaired student t-test. Asterisks are defining significance compared to the control group as: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. Intracellular loss of PKCδ in macrophages indicated IFN signaling and neutrophil migration at transcriptome level during Mtb infection.

[A] Experimental outline of bulk-RNA sequencing of BMDMs from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at indicated time points. [B-C] Volcano plots for uninfected (0 hr) and infected (24 hr) DEGs are shown between LysM^crePKCδ^flox/flox and PKCδ^flox/flox BMDMs. Upregulated and downregulated genes are defined as positive p-value and Log2 FC and negative p-value and Log2 FC, respectively. Dotted line denotes threshold (Fold change ≥ 0.5; p-value ≥ 0.05) on X- and Y- axis. [D] Horizontal dot plots (Ora) detailing the association of enriched Gene Ontology (GO) biological processes are shown between LysM^crePKCδ^flox/flox and PKCδ^flox/flox BMDMs at 24 hr post Mtb infection. [E] Heatmap of DEGs cluster associated with neutrophil migration. Significant DEGs are demarcated with doted square. [F] IREA cytokine enrichment plot showing the enrichment score (ES) for each of the cytokine response in LysM^crePKCδ^flox/flox BMDMs at 24 hr post Mtb infection. Bar length is representing the ES with darker red (enriched in LysM^crePKCδ^flox/flox BMDMs) and darker blue (enriched in PKCδ^flox/flox BMDMs). [G] Horizontal bar chart representing the top ranked transcription factors at 24 hr post Mtb infection according to their average integrated scores across all the libraries. All data shown are analysed and produced using R studio packages and appyters web-based software. Schematic cartoons by Biorender.com.

Figure 4.. Total- & Phospho- proteome analysis revealed dysregulated protein clusters contributing to neutrophil activation in PKCδ deficient macrophages during Mtb infection.

[A] Experimental outline of Total- and Phospho-proteome analysis of bone-marrow-derived macrophages (BMDMs) from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at indicated time points. [B-C] Heat-map analysis of total proteome of bone marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 0 minutes (Uninfected) and 30 minutes (MOI=1) post Mtb Infected (Infected) state. Protein clusters are arranged according to the similarity between their constituents. Horizontal and vertical clustering trees are depicting sample legends and differentially expressed proteins, respectively. [D] Gene Ontology (GO) analysis corresponding to differentially expressed proteins (DEP) shown in 0 min uninfected (B) and 30 min infected (C) group. The colour bars represents –log10(p-value) for the statistically significant top 10 biological processes. [E-F] Volcano plots for uninfected (0 min) and infected (30 min) phosphorylated DEP are shown between LysM^crePKCδ^flox/flox and PKCδ^flox/flox BMDMs. Upregulated, downregulated, and non-significant proteins are defined as red, green, and grey dots, respectively. Dotted line denotes threshold on X- and Y- axis. [G] Association of phosphorylated DEP to top 10 Reactome pathway database is shown at 0 min uninfected and 30 min infected group. [H] Kinase enrichment analysis of significant phosphorylated DEP corresponding to 0 min uninfected (E) and 30 min infected (F) group. Bars represent the mean rank of the top 10 kinases based on multiple library databases (color-coded above). Schematic cartoons by Biorender.com.

Figure 5.. Exogenous GM-CSF and IFN signaling blockade independently restores the intrinsic protection in PKCδ deficient BMDMs against Mtb infection.

[A] Determination of mycobacterial burden in GM-CSF stimulated (50 ng/ml) bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice during Mtb infection at indicated time points. [B] Total ATP production rate and metabolic dependence of GM-CSF stimulated bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 24 hours post Mtb infection. [C] Determination of Mean Fluorescence Intensity (MFI) of macrophage phenotypic M1 and M2 marker by flow cytometry in GM-CSF stimulated bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox and PKCδ^flox/flox mice at 24 hours post Mtb infection. [D] Determination of mycobacterial burden in CPZ (10 μg/mL), IFNβ (500U/mL), GM-CSF (50 ng/ml) stimulated bone-marrow-derived macrophages from LysM^crePKCδ^flox/flox at 24 hours post Mtb infection. [E] Determination of mycobacterial burden in the scramble siRNA treated and PRKCD deficient human monocyte-derived macrophages at indicated time points. [F] Comparison between the mean fluorescence intensity (MFI) of HN878-GFP at 24 hpi and 72 hpi using Fiji (Image processing module). [G] mRNA expression of PRKCD in bronchoalveolar lavage (BAL) samples acquired from active TB patients and household contacts (HHC) (N= 10/arm). [H] Proposed model of PKCδ in regulation of immunomodulatory macrophage functions during Mtb infection. All data shown mean+/− SD and is representative of two independent experiments performed in triplicates or quadruplicates. Statistical analyses were performed using an unpaired student t-test. Asterisks are defining significance compared to the control group as: *p < 0.05, **p < 0.01, ****p < 0.0001. Schematic cartoons by Biorender.com.