Changes in Mycobacterium tuberculosis-Specific Immunity With Influenza co-infection at Time of TB Diagnosis

Abstract

Background: Prior Influenza A viral (IAV) infection has been shown to increase susceptibility to tuberculosis (TB) and TB has also been shown to be a primary cause of death during pandemics, including the Spanish Influenza outbreak of 1918-1919. The majority of data has been obtained from mouse models, thus the aim of this study was to determine the impact of Flu co-infection on host immunity and disease severity in TB patients at diagnosis. Methods: Sputum from 282 patients with active TB were analyzed for presence of FluA/FluB RNA at presentation using multiplex PCR. Sputum RNA was also analyzed for Mycobacterium tuberculosis (Mtb) load using ¹⁶S RNA amplification. Supernatants from digested sputum and Mtb antigen-stimulated whole blood were analyzed using multiplex cytokine arrays and PBMC were analyzed for cytokine production from CD4+ T, CD8+ T and Mucosal Associated Invariant T cells (MAITs). Results: 12 (4.3%) of TB patients were found to have FluA or FluB viral RNA present in their sputum at the time of TB diagnosis. The TB/Flu co-infected patients had a significantly higher bacterial load compared to those with TB mono-infection (p = 0.0026). They had lower levels of IL17A in ex vivo sputum (p = 0.0275) and higher MCP-1 (CCL2) levels in the blood following PPD stimulation (p = 0.0267). TB/Flu co-infected subjects had significantly higher IFN-γ+IL-17+CD4+ and IFN-γ+IL-17-CD8+ cells compared to TB mono-infected subjects. Conclusions: These data show that Flu co-infection at time of TB diagnosis is associated with a higher bacterial load and differential cellular and soluble profiles. These findings show for the first time the impact of TB/Flu co-infection in a human cohort and support the potential benefit of Flu vaccination in TB-endemic settings.

Keywords: bacterial load; cytokines; flow cytometry; influenza; tuberculosis.

Figure 1

TB/Flu co-infection significantly increases bacterial load in sputum at diagnosis. ¹⁶S rRNA analysis was performed on sputum samples from TB/Flu co-infected (n = 11) and TB mono-infected (n = 110) patients at baseline. Bacterial load was calculated from a standard curve using H37Rv reference strain. Data were analyzed using Mann-Whitney U-test. Bar indicates median.

Figure 2

Cytokine profiles in TB/Flu Co-infection. (A) heat map showing differences in 27 cytokines/chemokines in sputum (n = 11 co-infected and n = 135 mono-infected) and antigen-stimulated blood (n = 6 co-infected and n = 80 mono-infected) from TB/Flu co-infected (A) and TB-mono-infected (B) patients. (B) MCP-1 levels in whole blood PPD-stimulated supernatants and (C) IL-17 levels in ex vivo sputum (both pg/ml). Data were analyzed using Mann-Whitney U-test; bar indicates median.

Figure 3

Increased cellular cytokines in TB/Flu co-infection. IFN-γ and IL-17 production from (A) CD4+ and (B) CD8+ T cells following PMA stimulation. On the left are representative flow cytometry plots showing IFN-γ (y-axis) and IL-17 (x-axis). Top is TB mono-infected and bottom is TB-Flu co-infection. The graphs on the right show grouped analysis. Data were analyzed using Mann-Whitney U-test. Bar indicates median.

Figure 4

Cytokine production from MAIT cells. (A,B) Representative FACS plots showing CD4+CD161+Va7.2+ (A) and CD8+CD161+Va7.2+ (B) cells. (C,D) total CD4+ and CD8+ MAIT cells. (E,F) IL-17+ MAIT cells and (G,H) IFN-γ+ MAIT cells. Data were analyzed using Mann-Whitney U-test. Bar indicates median. Black dots = TB mono-infected subjects; white dots = TB/Flu co-infected subjects.