Mice infected with Mycobacterium tuberculosis are resistant to acute disease caused by secondary infection with SARS-CoV-2

Abstract

Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to the pathological consequences of secondary CoV2 infection, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

Fig 1. Mtb-infected ACE2 mice are resistant to secondary infection with CoV2.

(A) Experimental overview of our ACE2:CoV2 model studies, wherein mice were infected via aerosol with Mtb (Day -30) and challenged 30 days later (Day 0) with CoV2. On post-challenge Day 4, Day 7 and Day 14, tissues were collected for microbiological and immunological assessments. Experimental groups included ACE2 mice which were not infected with Mtb prior to CoV2 challenge (Mtb^NEGCoV2^POS), ACE2 mice which were infected with Mtb but challenged with sterile saline (Mtb^POSCoV2^NEG), ACE2 mice which were infected with Mtb prior to CoV2 challenge (Mtb^POSCoV2^POS), and B6 controls which were infected with Mtb (to determine what if any impact human ACE2 transgene expression alone has on Mtb burdens). (B) The percent weight change experienced by each group of ACE2 mice following CoV2 challenge, as normalized to the original weight of each mouse. (C) CoV2 PFU burdens and (D) CoV2 N protein levels in the lungs of Mtb^NEGCoV2^POS and Mtb^NEGCoV2^POS mice. (E-G) Mtb CFU burdens in the (E) lungs, (F) spleen and (G) liver of Mtb^POSCoV2^NEG and Mtb^POSCoV2^POS mice, as well as B6 controls throughout the experiment time course. In each graph the following legend applies: Mtb^NEGCoV2^POS, black circles or bars; Mtb^POSCoV2^NEG, gray circles; Mtb^POSCoV2^POS, white circles or bars. (H) Representative micrographs of AFB stained lung sections, as collected from Mtb^POSCoV2^NEG and Mtb^POSCoV2^POS mice at the indicated times post-challenge. In each micrograph, the large scale bar is 20 μM and inset scale bar is 5 μm. This experiment was repeated twice, each with similar results (4 mice/group/timepoint). *, p ≤ 0.05 as determined by either Student’s t-test or ANOVA; n.s., not significant.

Fig 2. CoV2-elicted cytokine responses are muted in the presence of Mtb infection.

On the indicated days, lung tissue from Mtb^NEGCoV2^POS, Mtb^POSCoV2^NEG, Mtb^POSCoV2^POS and uninfected (UI) ACE2 mice was used to measure (A-C) protein levels of (A) IFNγ, (B) IL6 and (C) IL1β, as well as (D-I) mRNA levels of (D) IFNγ, (E) TNFα, (F) IFIT2, (G) IFIT3, (H) CCL2 and (I) IL10. This experiment was repeated twice, each with similar results (4 mice/group/timepoint). *, p ≤ 0.05 as determined by either Student’s t-test or ANOVA; §, significant relative to UI protein levels.

Fig 3. CoV2 infection of the airways and associated pneumonia are attenuated in the presence of Mtb.

(A-B) Representative micrographs of H&E stained lung sections, sections from each experimental group, as collected on (A) Day 4 or (B) Day 7 post challenge. (C-D) Quantitative analysis of H&E stains demonstrating (C) the size of each individual TB granuloma relative to the total lung area, with each dot representing an individual TB granuloma on the indicated Days, as well as (D) the cumulative area taken up by all granulomas in an individual mouse lung, with each dot representing an individual mouse on the indicated Days. Bars represent the mean +/- SD. (E-F) Representative micrographs of CoV2 N protein IHC stained lung sections from each experimental group, as collected on (E) Day 4 or (F) Day 7 post challenge. In each micrograph the large scale bar represents 200 microns; insets are 50 microns.

Fig 4. Mtb-infected B6 mice are resistant to secondary infection with MACoV2.

(A) Experimental overview of our B6:MACoV2 model studies, wherein mice were infected via aerosol with Mtb (Day -30) and challenged 30 days later (Day 0) with MACoV2. On post-challenge Days 4, 7 and14 we collected lung tissue for microbiological and immunological assessments. Experimental groups included B6 mice which were not infected with Mtb prior to MACoV2 challenge (Mtb^NEGMACoV2^POS), B6 mice which were infected with Mtb but challenged with sterile saline (Mtb^POSMACoV2^NEG), and B6 mice which were infected with Mtb prior to CoV2 challenge (Mtb^POSMACoV2^POS). (B) The percent weight change experienced by each group of B6 mice following MACoV2 challenge, as normalized to the original weight of each mouse. (C-D) Lung viral burdens in Mtb^NEGMACoV2^POS and Mtb^POSMACoV2^POS mice, as measured by (C) MACoV2 PFU or (D) MACoV2 N protein concentration on the indicated days, as well as (E) lung Mtb CFU burdens at the same timepoints. (F, H) Lung protein levels of (F) IFNγ and (H) IL6, as well as (G, I-K) mRNA levels of (G) IFNγ, (I) IFIT3, (J) IFITM3 and (K) ACE2. This experiment was repeated twice, each with similar results (4 mice/group/timepoint). *, p ≤ 0.05 as determined by either Student’s t-test or ANOVA; §, significant relative to UI protein levels.

Fig 5. Lung T and B cell subsets expand upon challenge of Mtb^POS mice with MACoV2.

As an unbiased means to define and compare the lung immune landscape, live CD45+ cells were purified from the lungs of four experimental groups (Uninfected, UI; Mtb^NEGMACoV2^POS; Mtb^POSMACoV2^NEG; Mtb^POSMACoV2^POS) on post-challenge Day 7 and used for scRNA analysis. (A-C) t-SNE plots of the resulting data, either (A-B) pooled across groups or (C) segregated by group to show (A) the extent of overlay and (B-C) clustering of data into 12 immune lineages. (D) The distribution and expression patterns of lineage defining genes which we used to annotate each cluster, as pooled from all group data (individual group data are shown in S1 Fig). (E) The proportion of each immune lineage in the lungs of each experimental group. MØ, macrophage; DC, dendritic cell; NK, natural killer.