Multi-layered transcriptomic analyses reveal an immunological overlap between COVID-19 and hemophagocytic lymphohistiocytosis associated with disease severity

Authors: Lena F. Schimkea,5, Alexandre H.C. Marquesa, Gabriela Crispim Baiocchia, Caroline Aliane de Souza Pradob Dennyson Leandro M. Fonsecab , Paula Paccielli Freirea , Desirée Rodrigues Plaçab , Igor Salerno Filgueirasa ,Ranieri Coelho Salgadoa, Gabriel Jansen-Marquesc, Antonio Edson Rocha liveirab
, Jean PierreSchatzmann Perona, José Alexandre Marzagão Barbutoa,d, Niels Olsen Saraiva Camaraa
, Vera Lúcia Garcia Calicha , Hans D. Ochse, Antonio Condino-Netoa, Katherine A. Overmyerf,g, Joshua J. Coonh,i, JosephBalnisj,k, Ariel Jaitovichj,k, Jonas Schulte-Schreppingl, Thomas Ulasm, Joachim L. Schultzel,m, Helder I.Nakayab, Igor Jurisican,o,p, Otavio Cabral-Marquesa,b,q

Clinical and hyperinflammatory overlap between COVID-19 and hemophagocytic lymphohistiocytosis (HLH) has been reported. However, the underlying mechanisms are unclear. Here we show that COVID-19 and HLH have an overlap of signaling pathways and gene signatures commonly dysregulated, which were defined by investigating the transcriptomes of
1253 subjects (controls, COVID-19, and HLH patients) using microarray, bulk RNA-sequencing (RNAseq), and single-cell RNAseq (scRNAseq). COVID-19 and HLH share pathways involved in cytokine and chemokine signaling as well as neutrophil-mediated immune responses that associate with COVID-19 severity. These genes are dysregulated at protein level across several
COVID-19 studies and form an interconnected network with differentially expressed plasma proteins which converge to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils.

Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.

More than one year of Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome Coronavirus (SARS-CoV)-2, more than 197 million cases and 4,2 million deaths have been reported worldwide (July 30th 2021, WHO COVID-19 Dashboard). The clinical presentation ranges from asymptomatic to severe disease manifesting as pneumonia, acute respiratory distress syndrome (ARDS), and a life-threatening hyperinflammatory syndrome associated with excessive cytokine release (hypercytokinaemia)1–3 . Risk factors for severe manifestation and higher mortality include old age as well as hypertension, obesity, and diabetes4. Currently, COVID-19 continues to spread, new variants of SARS-CoV-2 have been reported and the number of infections resulting in death of young individuals with no comorbidities has increased the mortality rates among the young population 5,6. In addition, some novel SARS-CoV-2 variants of concern appear to escape neutralization by vaccine-induced humoral immunity7 . Thus, the need for a better understanding of the immunopathologic mechanisms associated with severe SARS-CoV-2 infection.

Patients with severe COVID-19 have systemically dysregulated innate and adaptive immune responses, which are reflected in elevated plasma levels of numerous cytokines and chemokines including granulocyte colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), interleukin (IL)-6, IL-6R, IL18, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 10
(CXCL10)8–10 , and hyperactivation of lymphoid and myeloid cells11. Notably, the hyperinflammation in COVID-19 shares similarities with cytokine storm syndromes such as those triggered by sepsis, autoinflammatory disorders, metabolic conditions and malignancies12–14 ,often resembling a hematopathologic condition called hemophagocytic lymphohistiocytosis
(HLH)15. HLH is a life-threatening progressive systemic hyperinflammatory disorder characterized by multi-organ involvement, fever flares, hepatosplenomegaly, and cytopenia due to hemophagocytic activity in the bone marrow15–17 or within peripheral lymphoid organs such as pulmonary lymph nodes and spleen. HLH is marked by aberrant activation of B and T lymphocytes and monocytes/macrophages, coagulopathy, hypotension, and ARDS. Recently, neutrophil hyperactivation has been shown to also play a critical role in HLH development18,19. This is in agreement with the observation that the HLH-like phenotype observed in severe COVID-19 patients is due to an innate neutrophilic hyperinflammatory response associated with available under aCC-BY-NC-ND 4.0 International license. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

It is made bioRxiv preprint doi: ttps://; this version posted August 1, 2021. The copyright holder for this preprint
virus-induced hypercytokinaemia which is dominant in patients with an unfavorable clinical course17 . Thus, HLH has been proposed as an underlying etiologic factor of severe COVID191,3,20. HLH usually develops during the acute phase of COVID-191,20–27 . However, a case of HLH that occurred two weeks after recovery from COVID-19 has recently been reported as the cause
of death during post-acute COVID-19 syndrome28
The familial form of HLH (fHLH) is caused by inborn errors of immunity (IEI) in different genes encoding proteins involved in granule-dependent cytotoxic activity of leukocytes such as AP3B1, LYST, PRF1, RAB27A, STXBP2, STX11, UNC13D29–31. In contrast, the secondary form (sHLH) usually manifests in adults following a viral infection (e.g., adenovirus, EBV, enterovirus, hepatitis viruses, parvovirus B19, and HIV)32,33, or in association with autoimmune /rheumatologic, malignant, or metabolic conditions that lead to defects in T/NK cell functions and excessive inflammation16,31. fHLH and sHLH affect both children and adults, however, the clinical and genetic distinction of HLH forms is not clear since immunocompetent children can develop sHLH 34,35, while adult patients with sHLH may also have germline mutations in HLH genes36. Of note, germline variants in UNC13D and AP3B1 have also been
identified in some COVID-19 patients with HLH phenotype37, thus, indicating that both HLH forms may be associated with COVID-19.

Here, we characterized the signaling pathways and gene signatures commonly dysregulated in both COVID-19 and HLH patients by investigating the transcriptomes of 1253 subjects (controls, COVID-19, and HLH patients) assessed by microarray, bulk RNA-sequencing (RNAseq), and single-cell RNAseq (scRNAseq) (Table 1). We found shared gene signatures and cellular signaling pathways involved in cytokine and chemokine signaling as well as neutrophilmediated immune responses that associate with COVID-19 severity.

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Pre-existing immunity to SARS-CoV-2: the knowns and unknowns

Authors: Alessandro Sette 1 2Shane Crotty 3 4


T cell reactivity against SARS-CoV-2 was observed in unexposed people; however, the source and clinical relevance of the reactivity remains unknown. It is speculated that this reflects T cell memory to circulating ‘common cold’ coronaviruses. It will be important to define specificities of these T cells and assess their association with COVID-19 disease severity and vaccine responses.

As data start to accumulate on the detection and characterization of SARS-CoV-2 T cell responses in humans, a surprising finding has been reported: lymphocytes from 20–50% of unexposed donors display significant reactivity to SARS-CoV-2 antigen peptide pools1,2,3,4.

In a study by Grifoni et al.1, reactivity was detected in 50% of donor blood samples obtained in the USA between 2015 and 2018, before SARS-CoV-2 appeared in the human population. T cell reactivity was highest against proteins other than the coronavirus spike protein, but T cell reactivity was also detected against spike. The SARS-CoV-2 T cell reactivity was mostly associated with CD4+ T cells, with a smaller contribution by CD8+ T cells1. Similarly, in a study of blood donors in the Netherlands, Weiskopf et al.2 detected CD4+ T cell reactivity against SARS-CoV-2 spike peptides in 1 of 10 unexposed subjects and against SARS-CoV-2 non-spike peptides in 2 of 10 unexposed subjects. CD8+ T cell reactivity was observed in 1 of 10 unexposed donors. In a third study, from Germany, Braun et al.3 reported positive T cell responses against spike peptides in 34% of SARS-CoV-2 seronegative healthy donors. Finally, a study of individuals in Singapore, by Le Bert et al.4, reported T cell responses to nucleocapsid protein nsp7 or nsp13 in 50% of subjects with no history of SARS, COVID-19, or contact with patients with SARS or COVID-19. A study by Meckiff using samples from the UK also detected reactivity in unexposed subjects5. Taken together, five studies report evidence of pre-existing T cells that recognize SARS-CoV-2 in a significant fraction of people from diverse geographical locations.

These early reports demonstrate that substantial T cell reactivity exists in many unexposed people; nevertheless, data have not yet demonstrated the source of the T cells or whether they are memory T cells. It has been speculated that the SARS-CoV-2-specific T cells in unexposed individuals might originate from memory T cells derived from exposure to ‘common cold’ coronaviruses (CCCs), such as HCoV-OC43, HCoV-HKU1, HCoV-NL63 and HCoV-229E, which widely circulate in the human population and are responsible for mild self-limiting respiratory symptoms. More than 90% of the human population is seropositive for at least three of the CCCs6. Thiel and colleagues3 reported that the T cell reactivity was highest against a pool of SARS-CoV-2 spike peptides that had homology to CCCs.

What are the implications of these observations? The potential for pre-existing crossreactivity against COVID-19 in a fraction of the human population has led to extensive speculation. Pre-existing T cell immunity to SARS-CoV-2 could be relevant because it could influence COVID-19 disease severity. It is plausible that people with a high level of pre-existing memory CD4+ T cells that recognize SARS-CoV-2 could mount a faster and stronger immune response upon exposure to SARS-CoV-2 and thereby limit disease severity. Memory T follicular helper (TFH) CD4+ T cells could potentially facilitate an increased and more rapid neutralizing antibody response against SARS-CoV-2. Memory CD4+ and CD8+ T cells might also facilitate direct antiviral immunity in the lungs and nasopharynx early after exposure, in keeping with our understanding of antiviral CD4+ T cells in lungs against the related SARS-CoV7 and our general understanding of the value of memory CD8+ T cells in protection from viral infections. Large studies in which pre-existing immunity is measured and correlated with prospective infection and disease severity could address the possible role of pre-existing T cell memory against SARS-CoV-2.

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