Discussion:
In this study we describe cellular immune signatures characterizing mild (asymptomatic as well as symptomatic) and hypoxemic (moderate and severe) COVID-19 disease. We further elucidate the role of sMAdCAM, an important mucosal and lymphocyte migration marker and IL-15, a cytokine regulating leukocyte activation and homeostasis, in the observed cellular immune signatures.
Our results clearly illustrate, as has been well described (13–16), leukopenic profiles only in hypoxemic COVID-19 affected individuals. Further, in-depth analysis of T cell subsets revealed distinct depletion profiles in operation within CD4+ and CD8+ T cell compartments. While the CD4+ T cell compartment did not show any preferential depletion across naïve and memory subsets, we report here an asymmetric depletion profile in hypoxemic COVID-19 infection characterized by drastic depletion of naïve CD8+ T cells with relative sparing of the CD8+ effector memory T cell subset. These results extend the limited data available for T cell subsets in a stratified manner to include distinct stages of COVID-19 pathogenesis (10, 17). The CD4+ T regulatory cell subset represents a critical part of the response to viral infections (18, 19) that may play both protective as well as exacerbatory roles in COVID-19 pathogenesis. Our analysis revealed an incremental decrease in absolute count of this subset from moderate to severe pathogenesis compared to healthy controls. However, both stages of hypoxemic infection showed a relative preservation of this subset evidenced through elevated frequencies compared to healthy controls. As has been reported very recently this elevated frequency may represent suppressive (protective) as well as inflammatory roles played by these cells, markedly higher, in severe disease (20). Our study also evaluated the non-lymphocyte compartment in terms of monocytes and their profiles in mild and hypoxemic COVID-19 disease. This compartment has been known to modulate inflammatory responses to a variety of viral infections (21, 22). Interestingly, as opposed to the T cell compartment and when compared with healthy seronegative individuals, a pan-disease signature was delineated within monocytes comprising of elevated frequencies of intermediate monocytes. Also, concomitant decrease in frequency of non-classical monocytes was observed in mild infection irrespective of symptoms.
In an effort to understand factors influencing the observed cellular depletion and dysfunction in our cohort we evaluated plasma markers that could influence inflammation, mucosal leukocyte migration and homeostasis. Extending and strengthening our previous results on the converse relationship between sMAdCAM and IL-6 observed in non-hypoxemic COVID-19 progression (12), we report a similar and inversely correlated relationship between sMAdCAM and IL-6 levels across mild and hypoxemic stages of COVID-19 disease. Decreased sMAdCAM levels observed following SARS-CoV-2 infection in our study complement recently reported findings of reduced circulating lymphocytes expressing integrin α4β7 in peripheral blood of COVID-19 patients. Furthermore, the restoration of both sMAdCAM levels (in our study) and α4β7 expressing lymphocytes (23) was observed in convalescent individuals. Taken together, these results highlight a clear role for altered mucosal homing in the pathogenesis of this disease Furthermore, for the first time, we show that this converse relationship extends to opposing correlations with observed cellular immune signatures defining COVID-19 pathogenesis. Notably, elevated circulating LPS (a marker for gut inflammation), in the context of SARS-CoV-2 infection, has only been reported for severe and ICU patients (24). In our study we also observed increased plasma LPS levels in hypoxemic individuals compared to seronegative controls as opposed to our previous work with a cohort of mild COVID-19 (12). Importantly, there was no correlation between circulating LPS levels and either sMAdCAM or IL-6 levels. These results suggest that the altered trajectory of sMAdCAM levels is less related to gut persistence mediated inflammation and more to altered mucosal leukocyte migration. Taken together, our results show that IL-6 and sMAdCAM may contribute towards disparate aspects of disease progression where circulating levels of the former are more closely related to inflammatory sequalae and development of anti-viral B cell responses (25, 26) and those of the latter with restoration of mucosal homeostasis of leukocytes. Indeed, we have recently highlighted a role for the modulation of sMAdCAM and expression of its receptor integrin alpha-4 beta-7 present on monocytes, lymphocytes in the context of HIV pathogenesis (27).
While most immune markers of pathogenesis in COVID-19 have been reported to be associated with mild, moderate and severe disease, a marker that clearly discriminates between symptomatic and asymptomatic pathogenesis remains elusive. Our study is the first to demonstrate the presence of plasma IL-15 as such a marker where detection of this cytokine is clearly segregated based on the symptomatic status of patients with incrementally higher levels detected in mild symptomatic to hypoxemic individuals. In fact, of the 2 (out of 11) mild asymptomatic individuals that had detectable levels of this cytokine, one was found retrospectively to have progressed to a hypoxemic stage. Also, negative correlation of plasma IL-15 levels with lymphopenic profiles (T, B, NK) are indicative of its homeostatic restorative production (28–30) in the face of systemic depletion of these subsets. Circulating IL-15 levels have been implicated as a contributory factor to hospitalization time, disease severity and mortality in some settings (31, 32). Indeed, a preliminary analysis in our cohort, with respect to days of hospitalization supported this finding. Our study further demonstrates the utility of using it as an early biomarker predicting progression to symptomatic and severe disease.