Melissa Govender

SARS-CoV-2 infection remains a global health concern, with its impact on host immune responses not fully understood. In a case–control study, we examined how COVID-19 affects DNA methylation patterns in the upper respiratory airway of hospitalized individuals. DNA methylation arrays were performed on nasopharyngeal samples at inclusion/hospitalization and 6 weeks post-inclusion. We found a distinct DNA methylation pattern in COVID-19 patients compared to healthy controls, identifying 510,099 differentially methylated CpGs. Within the transcription start sites (TSSs) and gene body, COVID-19 patients displayed a higher number of genes/CpGs with elevated methylation levels. Enrichment analysis of TSS-methylated genes revealed effects of SARS-CoV-2 on genes associated with type I interferons, anti-viral and inflammatory responses, and immune functions. Some CpG methylations were transient, and normalized at group level by 6 weeks post-inclusion. Several IFN-regulated genes, including OAS1, OAS3, IFIT3, and MX1, were identified. Among the top regulators were IL17A and ERK1/2, both involved in inflammatory processes. Networks nodes included IGF1 and EGF, associated with processes including tissue repair and activation of immune responses. Overall, our data suggests that COVID-19 can impact the upper airway by modifying gene methylation patterns. This could have implications for conditioning of the airways, how individuals respond to future airway infections, and therapeutic interventions.

The emergence of SARS-CoV-2 has had a profound adverse impact on global health and continues to remain a threat worldwide. The disease spectrum of COVID-19 ranges from asymptomatic to fatal clinical outcomes especially in the elderly population and in individuals with underlying medical conditions. The impact of COVID-19 on host immune responses and immune cells at the protein and DNA levels remains largely ambiguous. In a case-control study, here we explored the impact of COVID-19 on DNA methylation patterns in the upper respiratory airway to determine how SARS-CoV-2 infection altered the immune status of individuals requiring hospitalization for COVID-19. We performed DNA methylation arrays on nasopharyngeal swabs at inclusion/hospitalization as well as 6 weeks post-inclusion. Our study reveals a distinct DNA methylation pattern in COVID-19 patients compared to healthy controls, characterized by 317 779 differentially methylated CpGs. Notably, within the transcription start sites and gene body, COVID-19 patients exhibited a higher number of genes/CpGs with elevated methylation levels. Enrichment analysis of methylated genes at transcription start sites highlighted the impact on genes associated with inflammatory responses and immune functions. Some SARS-CoV-2 -induced CpG methylations were transient, returning to normal levels by 6 weeks post-inclusion. Enriched genes of interest included IL-17A, a pivotal cytokine implicated with inflammation and healing, and NUP93, associated with antiviral innate immunity. Further, six genes in our data set, OAS1, CXCR5, APP, CCL20, CNR2, and C3AR1, were found in enrichment analysis with previous COVID-19 studies. Additionally, RNAse1 and RNAse2 emerged as key regulators, while IL-18 played a role in various biological processes in COVID-19 patients. Overall, our results demonstrates that COVID-19 has a major impact on the upper airway by modifying the methylation pattern of many genes and this could have implications for the conditioning of the airways and how the individual response to future airway infections.

After more than two years the COVID-19 pandemic continues to burden healthcare systems and economies worldwide, and it is evident that long-term effects of the disease can persist for months post-recovery in some individuals. The activity of myeloid cells such as monocytes and dendritic cells (DC) is essential for correct mobilization of the innate and adaptive responses to a pathogen. Impaired levels and responses of monocytes and DC to SARS-CoV-2 is likely to be a driving force behind the immune dysregulation that characterizes severe COVID-19. Here, we followed, for 6-7 months, a cohort of COVID-19 patients hospitalized during the early waves of the pandemic. The levels and phenotypes of circulating monocyte and DC subsets were assessed to determine both the early and long-term effects of the SARS-CoV-2 infection. We found increased monocyte levels that persisted for 6-7 months, mostly attributed to elevated levels of classical monocytes. While most DC subsets recovered from an initial decrease, we found elevated levels of cDC2/cDC3 at the 6-7 month timepoint. Analysis of functional markers on monocytes and DC revealed sustained reduction in PD-L1 expression but increased CD86 expression across almost all cell types examined. Finally, viral load and CRP correlated to the appearance of circulating antibodies and levels of circulating DC and monocyte subsets, respectively. By elucidating some of the long-term effects that SARS-CoV-2 infection has on these key innate myeloid cells, we have shed more light on how the immune landscape remains affected in the months following severe COVID-19.

COVID-19 is being extensively studied, and much remains unknown regarding the long-term consequences of the disease on immune cells. The different arms of the immune system are interlinked, with humoral responses and the production of high-affinity antibodies being largely dependent on T cell immunity. Here, we longitudinally explored the effect COVID-19 has on T cell populations and the virus-specific T cells, as well as neutralizing antibody responses, for 6-7 months following hospitalization. The CD8 ⁺ TEMRA and exhausted CD57 ⁺ CD8 ⁺ T cells were markedly affected with elevated levels that lasted long into convalescence. Further, markers associated with T-cell activation were upregulated at the inclusion, and in the case of CD69 ⁺ CD4 ⁺ T cells this lasted all through the study duration. The levels of T cells expressing negative immune checkpoint molecules were increased in COVID-19 patients and sustained for a prolonged duration following recovery. Within 2-3 weeks after symptom onset, all COVID-19 patients developed anti-nucleocapsid IgG and spike-neutralizing IgG as well as SARS-CoV-2-specific T cell responses. In addition, we found alterations in follicular T helper (TFH) cell populations, such as enhanced TFH-TH2 following recovery from COVID-19. Our study revealed significant and long-term alterations in T cell populations and key events associated with COVID-19 pathogenesis.

The skin microenvironment at the site of infection plays a role in the early events that determine protective T helper 1/type 1 immune responses during cutaneous leishmaniasis (CL) infection. During CL in nonhealing BALB/c mice, early interleukin-4 (IL-4) can instruct dendritic cells for protective Th1 immunity. Additionally, keratinocytes, which are the principal cell type in the skin epidermis, have been shown to secrete IL-4 early after Leishmania major infection. Here, we investigated whether IL-4/IL-13 signaling via the common IL-4 receptor alpha chain (IL-4Rα) on keratinocytes contributes to susceptibility during experimental CL. To address this, keratinocyte-specific IL-4Rα-deficient (KRT14^cre IL-4Rα^−/lox) mice on a BALB/c genetic background were generated by gene targeting and site-specific recombination (Cre/loxP) under the control of the keratinocyte-specific krt14 locus. Following high-dose infection with L. major IL-81 and LV39 promastigotes subcutaneously in the footpad, footpad swelling, parasite burden, IFN-γ/IL-4/IL-13 cytokine production, and type 1 and type 2 antibody responses were similar between KRT14^cre IL-4Rα^−/lox and littermate control IL-4Rα^−/lox BALB/c mice. An intradermal infection with low-dose L. major IL-81 and LV39 promastigotes in the ear showed results in infected KRT14^cre IL-4Rα^−/lox BALB/c mice similar to those of littermate control IL-4Rα^−/lox BALB/c mice, with the exception of a significant decrease observed in parasite burden only at the site of LV39 infection in the ear. Collectively, our results show that autocrine and paracrine signaling of IL-4/IL-13 through the IL-4Rα chain on keratinocytes does not influence the establishment of a nonhealing Th2 immune response in BALB/c mice during L. major infection.

Experimental infection with the protozoan parasite Leishmania major has been extensively used to understand the mechanisms involved in T helper cell differentiation. Following infection, C57BL/6 mice develop a small self-healing cutaneous lesion and they are able to control parasite burden, a process linked to the development of Th1 cells. The local presence of IL-12 has been reported to be critical in driving Th1 cell differentiation. In addition, the early secretion of IL-4 was reported to potentially contribute to Th1 cell differentiation. Following infection with L. major, early keratinocyte-derived IL-4 was suggested to contribute to Th1 cell differentiation. To investigate a putative autocrine role of IL-4 signaling on keratinocytes at the site of infection, we generated C57BL/6 mice deficient for IL-4Rα expression selectively in keratinocytes. Upon infection with L. major, these mice could control their inflammatory lesion and parasite load correlating with the development of Th1 effector cells. These data demonstrate that IL-4 signaling on keratinocytes does not contribute to Th1 cell differentiation. To further investigate the source of IL-4 in the skin during the first days after L. major infection, we used C57BL/6 IL-4 reporter mice allowing the visualisation of IL-4 mRNA expression and protein production. These mice were infected with L. major. During the first three days after infection, skin IL-4 mRNA expression was observed selectively in mast cells. However, no IL-4 protein production was detectable locally. In addition, early IL-4 blockade locally had no impact on subsequent Th1 cell differentiation and control of the disease. Taken together, the present data rule out a major role for skin IL-4 and keratinocyte IL-4Rα signaling in the development of a Th1 protective immune response following experimental infection with L. major.

Significance: Cutaneous leishmaniasis and schistosomiasis are neglected tropical diseases for which there are no effective vaccines and limited treatment strategies. To develop vaccine and therapeutic alternatives, a detailed understanding of host immunity is essential. We show a role for IL-4Rα–responsive B cells in host susceptibility toLeishmania majorand protection againstSchistosoma mansoniinfection through the production of early IL-4, which in turn regulates Th2 cell polarization and disease outcome in mice. These important findings highlight the significant impacts that B cell-specific IL-4Rα and IL-4 responsiveness have in the context of type 1 (L. major) and type 2 (S. mansoni) pathogens. Thus, vaccine and therapeutic development should aim to target both B and T cell immunity for optimal efficacy.