Unique genes: the innate immune response and tuberculosis


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Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis (Mtb), is a severe chronic disease that can affect humans and animals. Mtb is a leading cause of death in the world (1.5 million deaths every year). Among people who are infected, only 10% will develop TB, 90% of these infections are latent (LTBI). This pathology presents the difference in immune control of Mtb. Macrophages (MΦs) are significant phagocytes that play a key role in innate and adaptive immunity. Their phenotypes stimulated with IFNy (pro-inflammatory) or IL-4, IL-13, and IL-10 (anti-inflammatory) determine the type of Mtb response.

In a recent paper, Khan, et al., hypothesized that the MΦ phenotype can enhance anti-TB function following cytokine stimulation, and this stimulation can explain the development of LTBI (Figure 1). They showed that infected M1-MФs survived better with upregulation of genes and pathways that regulate antigen processing Compared to M2-MФs. They highlight also M1-MΦs driven with IFN-γ increased the ability to bacterial control with the pro-inflammatory cytokine, nitric oxide expression, and autophagy-dependent degradation of Mtb, whereas IL-4 programmed M2-polarized MΦs (M2-MΦs) are permissive for Mtb proliferation.

Figure 1: Human umbilical cord and peripheral blood-derived macrophages show heterogeneity in mycobacterial killing associated with oxidants and autophagy. Human cord blood (CBM) or healthy donor PBMC-derived MФs were cultured in the presence of either recombinant human IFN-γ (M1; 10 ng/mL) or human IL-4 (M2; 10 ng/mL) for 5 days and rested for 2 days. Untreated cells were M0-MФs. a, b Surface expression of receptors by CBM- derived naïve and Mycobacterium tuberculosis (Mtb; H37Rv)-infected M1- (CD80+/CD206−) and M2-MФs (CD80−/CD206+) on day 3 using flow cytometry and quantitation (*p < 0.01 t test); gating strategy shown Supplemental Fig. 1. c CBM-derived differentiated MФs were infected with Mtb for 4 h followed by microscopic counts of rfp-labeled MtbH37Rv to determine uptake verified using CFU counts. d PBMC-derived MФs from five healthy donors were differentiated using the indicated cytokines followed by infection with Mtb and CFU assay on day 4. Each point represents one donor (**p < 0.05; Kruskal–Wallis test). e PBMC-derived MФs were differentiated using GM-CSF (M1), IL-4 (M2a), IL-1β M2b), IL-10 (M2c) or left intreated (M0) followed by Mtb infection and CFU assay on day 4 (**p < 0.007). f CBM-derived, cytokine differentiated MФs were infected with Mtb followed by CFU assay over time (**p < 0.006; data from 3 experiments shown). g CBM-derived, differentiated MФs were infected with M. bovis BCG followed by CFU assay on day 4 (**p < 0.005). h CBM-derived and differentiated uninfected MФs or those infected with Mtb or BCG were incubated and at indicated time points, cultures were tested for nitrite using diaminofluorescein diacetate and fluorometry (*,**p < 0.005, t test). QPCR for mRNA of iNOS and protein are shown in Supplemental Fig. 3a and reactive oxygen species levels in Supplementary Fig. 3b. i MФs infected with Mtb as in panel h were incubated in NMMA (0.5 mM; N-monomethyl L-arginine) followed by CFU assay on day 3 (**p < 0.009). j CBM-derived, differentiated MФs infected with Mtb were incubated with 10 μM Rapamycin, 100 μM Metformin or their combination followed by CFU assay on day 3 (**p < 0.009). k CBM-derived, M1-, M2- and M0-MΦs were treated in the presence or absence of siRNA vs. beclin1 (ATG6) or its scrambled control followed by infection with Mtb and CFU counts on day 3 (*p < 0.007). Blot validation of Knockout using siRNA vs. beclin1 (ATG6) is shown in Fig. 3h. l MΦ lysates of panel k collected at 18 h were analyzed using western blots for the lipidation of microtubule-associated light chain 3 (LC3). Lipidation is indicated by LC3-II. m CBM-derived MΦs were infected with rfpMtbH37Rv and stained for an LC3 autophagy marker or LAMP1 lysosome marker using specific antibodies, Alex-Fluor485 conjugates, and imaged using confocal microscopy. Panels illustrate LC3 colocalization; LAMP1 stains using gfpMtbH37Rv is shown in Supplementary Fig. 3c. n Quantification of phagosomes colocalizing with LC3 are shown using an N90 Nikon fluorescence microscope (IF) and Metaview software (*p < 0.004, t test). For panels (b–c–e–f–g–h–i–b–k–n), p-values were calculated using a one-way ANOVA with Tukey’s post-hoc test; one of 2–3 similar experiments shown. CFU or IL-2 assays had triplicate wells plated per group or donor. Panels (d, g, i–k) horizontal dotted lines indicate day 0 CFU (4 h post-infection CFU). All Mtb CFU experiments used MOI of 1.

They found that positive autophagy regulation in Mtb-infected M1-MΦs strongly expressed Sirtuin5, HDAC2 (positive autophagy regulator). However, the expression of Sirtuin2 (negative autophagic regulator) Mtb-infected M2-MΦs was correlated to bacterial survival. They finally suggested that M1 and M2 phenotypes can mediate anti-TB response in humans and macaques. The gene expression difference could be used as Host directed therapeutic targeting MΦs polarization.

Journal article: Khan, A. et al., 2022. Human M1 macrophages express unique innate immune response genes after mycobacterial infection to defend against tuberculosis. Communications biology.

Summary by Mberkadji Ngana

 

 
 
 
 
 
 
International Union of Immunological SocietiesUniversity of South AfricaInstitute of Infectious Disease and Molecular MedicineElizabeth Glazer Pediatric Aids FoundationStellenbosch University