A new study published on the preprint server medRxiv* in June 2020 shows that the drug hydroxychloroquine (HCQ) suppresses a form of immunity called ‘trained immunity,’ with repercussions for its potential use to treat COVID-19.
HCQ is an anti-malarial and a disease-modifying anti-rheumatic drug (DMARD), which was observed to inhibit the replication of SARS-CoV-2 in vitro. However, its antiviral effect in humans in vitro has not been confirmed. It may have an immunomodulatory effect as well. However, the lack of evidence of its efficacy and how they act has roused controversy surrounding its use.
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HCQ Suppresses Trained Immunity
The current study aimed to understand how HCQ acts on the immune response in COVID-19. Using techniques that unravel the function of immune cells, as well as transcriptomic analyses, the researchers found marked changes in the expression of molecular markers and functionalities of monocytes in COVID-19 patients. They also found that interferon-stimulated genes (ISG) play a role in disease severity.
With metabolomic and epigenetic studies, the researchers found that HCQ suppresses trained immunity. Trained immunity refers to a change in the way that monocytes function in response to epigenetic changes that reprogram their antiviral responses. These findings suggest that HCQ may not be suitable for the therapy or prevention of COVID-19.
The Study of Monocytes and Associated Cytokines
The study included 13 patients hospitalized with SARS-CoV-2, all above 18 years of age. The median age was 68 years. They had various coexisting maladies such as pulmonary disease, cardiovascular disease, and malignancy.
Most patients were admitted to hospital with fever, cough, or breathlessness. Seven of the patients required oxygen at admission. All showed signs of pneumonitis on chest imaging, but none were critically ill. All patients were started on chloroquine (CQ) at admission, for five days.
Increase in Monocyte Count and Activation
On blood analysis, the T cell count was slightly lower than normal, and monocyte counts were markedly higher, mostly because of a rise in classical monocytes. Nonclassical monocytes were almost undetectable, and their markers, such as CX3CR1, were reduced.
HLA-DR expression on monocytes was reduced, which has been associated in recent studies with the hyperactivation of monocytes and the excessive release of the pro-inflammatory cytokine IL-6 in COVID-19. CD11b, a monocyte activation marker, is also upregulated. These markers remained constant over five days in those who were still hospitalized at the end of the study.
The researchers then examined the functional status of peripheral blood mononuclear cells (PBMCs) by stimulating them and then measuring the release of cytokines IL-1β, IL-6, and TNFα. They found that excessive cytokine release occurred in COVID-19 patients when lipopolysaccharides and other antigens were used to activate Toll-like receptor (TLR) 4 and other similar receptors.
Polarization of Adaptive Immunity
Next, they looked at whether adaptive immunity was also altered, by stimulating PBMCs for 7 days with Staphylococcal aureus antigens, and measuring Th1 and Th17 cell activation via IFNγ and IL-17 levels respectively. Healthy controls showed increases in the former alone, but in COVID-19 patients, the latter was raised. This indicates a shift towards Th17 cell activation rather than the normal Th1 dominance.
Predictors of Outcome
When they compared the 9 patients who recovered without intensive care unit (ICU) admission with the 4 who required ICU care or died (3 and 1, respectively), they found no clear markers at presentation to predict favorable or poor outcomes. However, immune markers showed differences such as a reduced monocyte HLA-DR expression in those who went on to poor outcomes, indicating a more severe inflammatory phenotype in monocytes for these patients.
Transcriptome analysis of monocytes from COVID-19 patients showed a higher level of transcription of ISG, which plays a significant role in antiviral responses. Excessively high ISG expression was linked to poor outcomes.
Six and seven patients were discharged within five days and remained hospitalized, respectively. PBMCs from this group at 5 days from admission showed a clear demarcation between findings indicating a good vs. poor outcome.
Specifically, the inflammatory response is characterized by marked innate immune changes, in agreement with previous reports, in the form of increased monocyte activation, increased ISG expression, and elevated monocyte-derived cytokine release.
The researchers comment, “This enhanced responsiveness is reminiscent of the inflammatory phenotype previously reported in sepsis and influenza. While inflammation early in the infection contributes to improved antiviral mechanisms and elimination of infection, if exacerbated late during the course of the disease it may play a role in the development of the severe complications of COVID-19.”
How HCQ Suppresses Trained Immunity
This led to an investigation into whether HCQ affects trained immunity. This molecule moves passively into the lysosomes and disrupts its function. Since the lysosomes are central to the regulation of immune metabolism with innate immunity via the mTOR receptor on its membrane, trained macrophages are characterized by the activation of key regulators of lysosome genes.
To understand how HCQ affects trained immunity, they repeatedly stimulated human PBMCs with bacterial antigens. They found that the cells produced much more cytokines with repeated stimulation, but this effect disappeared when the cells were treated with HCQ simultaneously.
On restimulation of monocytes with IFNγ, there was a rise in IL-6 and TNFα production, which also vanished with HCQ treatment. The researchers analyzed the change in terms of lysosomal function and found that the inhibition of vacuolar ATPase (V-ATPase) led to blocking the development of trained immunity, which is similar to the effect of HCQ treatment.
Next, the effects of hydroxychloroquine on the transcription of trained monocytes were analyzed. They found that this led to a substantial reduction in the expression of genes related to inflammatory responses, including ISG. This was accompanied by the increased expression of metabolic pathways involved in inflammation. Altogether, this implies the role of HCQ in suppressing the development of trained immunity and the expression of ISG.
HCQ also affects cellular lipid metabolism, as part of its suppressive effect on trained immunity. The stimulation of the monocytes by bacterial antigens, with and without HCQ exposure followed by lipidomic analysis, showed that the normal wide-ranging and deep changes in the monocyte lipids that accompanies trained immunity was suppressed by HCQ. This may affect the structure and function of the cellular membranes, disrupting the activity of membrane-bound genes, including the all-important mTOR on the lysosomal membrane, as well as inhibiting lipid-dependent activation enzymes required for the normal immune response.
HCQ also prevents the normal epigenetic modifications that are required for trained immunity, shutting down normal changes in epigenetic markers associated with immune and inflammatory responses.
Implications and Future Applications
The authors say this data provides crucial new information about how HCQ acts in COVID-19. Though HCQ has been used for decades as an immunomodulator to benefit rheumatoid arthritis and systemic lupus erythematosus because it inhibits pro-inflammatory cytokines like IL-6 and TNFα. It is known that this effect is partly mediated by its inhibition of lysosomal processes like autophagy, antigen processing, and TLR7 processing.
However, the current study adds to this knowledge via the findings that HCQ prevents the development of trained immunity via epigenetic regulation. This may be via its effect on mTOR signaling since this is a lysosome-associated enzyme transmitting information from the lysosome to the cell, and thus mediates inflammation. The data on the changes in lipids that play a key role in mTOR activation supports this reading.
Since trained immunity is required to upregulate the innate immune response and so prevent infection, HCQ is less likely to be of use in preventing or clearing SARS-CoV-2 infection. This agrees with the findings of a recent randomized controlled trial that HCQ given post-exposure does not help prevent symptomatic COVID-19.
The question remains whether the immunomodulatory effects of HCQ could mediate its effectiveness in severe COVID-19 by muting the cytokine storm. The researchers say this is likely to be less useful than IL-6 receptor antibodies or IL-1 receptor antagonists, and an observational study lends some support to this prediction. More research is required to test this hypothesis. They sum up: “Our findings suggest that hydroxychloroquine may not have a beneficial effect on the antiviral immune response in SARS-CoV-2 infection.”
medRxiv 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.
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