The ongoing COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has thrown much of the world into disarray, with over 4 million people reported infected and well over 282,000 deaths. A new study published on the preprint server bioRxiv* in May 2020 suggests that the long-term impact of the virus could include activation of dormant bacterial infections like tuberculosis (TB).
According to the World Health Organization (WHO), dormant TB already affects a quarter of the world’s population. If the novel coronavirus activates a sizable proportion of these dormant infections, it could severely upset the global health and economic situation. The current study aims at quantifying this association so as to shape policies that could help avert a global TB pandemic.
Many viruses, including SARS-CoV-2, cause a temporary immunosuppressive effect, which causes dormant bacterial infections to come back to life. This was the case with the Spanish flu pandemic of 1918-20, which caused an increase in the number of lung TB cases. The highest death rate was in the patient subgroup, which had influenza with TB.
The 2009 HIN1 flu pandemic also showed the same trend, with poorer outcomes in patients coinfected with TB or multidrug-resistant strains of TB (MDR-TB). Patients with SARS or MERS infections were also found to develop lung TB.
The researchers in the current study hypothesized that the CoV infections could be causing lung inflammation that leads to reactivation of dormant TB in the lung. Others say that both lung lesions and liver infection by the mycobacteria, which cause TB (Mycobacterium tuberculosis, MTB) are enhanced by the presence of influenza A in mouse models, which also show a type I interferon signaling pathway that increases mycobacterial growth.
The current study focuses on dormant TB in adult stem cells. These cells may live in the bone marrow as well as in inflamed areas. One subset, called CD271+BM-mesenchymal stem cells (CD271+BM-MSCs), could harbor the dormant mycobacteria in both mice and humans, allowing later reactivation.
In prior studies, the investigators developed a mouse model that shows how the stem cells mediate this phenomenon. Mice are infected with the mycobacteria and show granuloma formation as well as antibodies to these organisms, after being treated with streptomycin for 3 weeks. When they are later deprived of streptomycin for six months, the bacteria become non-replicating.
The MTB are found mostly within the CD271+MSCs of the bone marrow and the lung to a much lesser extent. Those in the bone marrow live under relatively hypoxic conditions.
It is known that the ability to culture MTB from non-CD271+MSCs in the lungs of these mice signal tuberculous reactivation, which makes this model useful in the study of stem cell-mediated MTB reactivation.
The researchers looked for proof of dormant MTB reactivation using a murine hepatitis virus strain-1 (MHV-1), which is a murine coronavirus capable of representing the clinical features of SARS-CoV-2 in humans. MHV-1 induces acute respiratory infection in 2-4 days of infection, which leads to acute lung inflammation, with high levels of inflammatory chemicals like TNF-alpha, within 2-14 days of infection, and then recover.
An in vitro study showed the susceptibility of type II alveolar epithelial cells to MHV-1, which allows the use of these cells to identify an antiviral defense mechanism mediated by these altruistic stem cells (ASC).
The study shows the activation of the ASC-associated innate defense mechanism on MHV-1 infection. This mechanism has previously been found in embryonic stem cells (ESCs). MHV-1 may activate these specialized defense mechanisms within MTB-positive stem cells, leading to increased MTB proliferation in the lung and thus reducing the viral load.
The researchers also observed an unexpected finding: MTB reactivation is linked to an increase in the number of lung cells in the vicinity that survive, leading to an interest in the possible therapeutic application of this defense mechanism.
Did the MHV-1 infection cause MTB reactivation?
The researchers administered MHV-1 intranasally to mice with dormant MTB in these stem cells, along with streptomycin, which is required for this strain of MTB to replicate. They found that in both test and control groups, the viral titer increased rapidly over the first four weeks but then went down. However, the viral load after two weeks was 20 times less in the test than in control mice. In both groups, the TNF-alpha levels went up by 3-4 times. These results suggest that viral replication and immune activation occurred in both groups, but the viral load was less in the mice with dormant MTB infection.
Secondly, the researchers infected one group of mice with dormant stem cell MTB with MHV-1. Another group with dormant MTB within stem cells was the control. Both were treated with streptomycin. By the 8th day post-infection, there was a 6 to 7-fold rise in the number of viable MTBs in both groups. After that, on days 8-20, the number went up by 110-fold in the test group, while it decreased to half in the control group.
The MHV-1 infection reactivated and maintained MTB proliferation while on streptomycin, while the drug failed to do so in the control group. The overall increase in MTBs was 630-fold in the test group over three weeks.
In the third step, the researchers treated mice with dormant MTBs with dexamethasone or aminoguanidine, two immunosuppressive drugs that can reactivate dormant MTB. Both groups were also given streptomycin.
After a month, they found that there was a 3-4-fold rise in the number of viable MTBs in both groups. They compared this to the rise in viable MTBs following MHV-1 infection for one month and found the latter to be 400 times higher. This not only shows the superior reactivation potential of MHV-1 but the operation of other mechanisms than immunosuppression in this process.
The researchers extracted the CD271+MSCs to examine the rate of replication and extracellular MTB release from three groups of mice: a) with dormant stem cell MTBs b) with dormant MTBs and MHV-1 infection c) without MTBs but with MHV-1 infection. They found that mice with dormant MTBs and MHV-1 coinfection had a 12-fold rise in the stem cell population from day 0-8. Mice with MHV-1 infection alone also showed expansion of this population, but six times less. There was no expansion in the MTB group alone.
The MTB-MHV-1 mice had a 27-fold rise in the number of intracellular viable MTBs, but not in the extracellular number, until days 8-12, when there was a 40-fold rise. Lung cells in vivo showed the same trend, with a 15-fold rise in the extracellular MTB number. Thus MHV-1 infection induces a transient increase in the intracellular MTB population and its release into the extracellular space.
The investigators also found that MHV-1 infection caused the stem cells to be reprogrammed as altruistic stem cells, a phenotype induced by some invading pathogens “that threaten the integrity of the stem cells residing in their niches.” This is characterized by the expression of specific genes that increase “stemness,” and a rapid increase in the number of reprogrammed cells. These show high stem cell marker expression for two weeks and then activate the apoptosis protein p53 to differentiate or die.
This may explain the transient increase in the stem cell population as well as well-defined changes in cell markers that indicate increased stem cell characteristics. This was 2-3 times higher in the stem cells containing dormant MTBs and exposed to MHV-1 than in the group with MHV-1 infection alone.
This ASC-mediated protection of the lung cell against MHV-1 was observed, with the type II alveolar epithelial cells being protected 2-3 times more efficiently by the conditioned media of the MSCs.
The researchers comment on the link between the national policy of BCG (Bacille Calmette-Guerin) vaccination against tuberculosis and the lower mortality from COVID-19. They note the close similarity of BCG with MTB, which could indicate the former also enhanced the ASC-mediated antiviral defense mechanism. They say, “Our results could provide a novel explanation of BCG mediated host defense or antiviral mechanism against SARS-CoV2.”
In short, MHV-1 infection induces inflammation that activates MTBs within the lung stem cells and mobilizes more MTB-containing stem cells from the bone marrow to the lung. This reactivation triggers ASC behavior, which boosts antiviral defenses.
This phenomenon might also partially explain how the cytokine storm in COVID-19 patients treated with mesenchymal stem cells (MSCs) is reduced because MSCs are known to be immunosuppressive. The observed MSC to ASC conversion could further boost this immunomodulatory activity.
This model also offers an explanation for the mechanism of protection offered by BCG vaccination against florid COVID-19 symptoms, by triggering MSC to ASC conversion and enhancing immunomodulatory cytokine production.
The study may help understand “how MSCs inducing ASC defense mechanism will help in combating the viral load in the host, thereby helping in developing a possible cure for COVID-19.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.