Protective reactive thymus hyperplasia in COVID-19 acute respiratory distress syndrome.

BACKGROUND: Patients with COVID-19 (COVID) may develop acute respiratory distress syndrome with or without sepsis, coagulopathy and visceral damage. While chest CT scans are routinely performed in the initial assessment of patients with severe pulmonary forms, thymus involvement and reactivation have not been investigated so far. METHODS: In this observational study, we systematically scored the enlargement of the thymus and the lung involvement, using CT scans, in all adult patients admitted to the ICU for COVID or any other cause (control group) at one centre between March and April 2020. Initial biological investigations included nasal detection of SARS-CoV-2 ribonucleic acid by polymerase chain reaction (PCR). In a subgroup of 24 patients with different degrees of pulmonary involvement and thymus hypertrophy, plasma cytokine concentrations were measured and the export of mature T cells from the thymus was estimated simultaneously by PCR quantification of T cell receptor excision circles (TRECs). RESULTS: Eighty-seven patients were studied: 50 COVID patients and 37 controls. Non-atrophic or enlarged thymus was more commonly observed in COVID patients than in controls (66% vs. 24%, p < 0.0001). Thymus enlargement in COVID patients was associated with more extensive lung injury score on CT scans (4 [3-5] vs. 2 [1.5-4], p = 0.01), but a lower mortality rate (8.6% vs. 41.2%, p < 0.001). Other factors associated with mortality were age, lymphopaenia, high CRP and co-morbidities. COVID patients had higher concentrations of IL-7 (6.00 [3.72-9.25] vs. 2.17 [1.76-4.4] pg/mL; p = 0.04) and higher thymic production of new lymphocytes (sj/ßTREC ratio = 2.88 [1.98-4.51] vs. 0.23 [0.15-0.60]; p = 0.004). Thymic production was also correlated with the CT scan thymic score (r = 0.38, p = 0.03) and inversely correlated with the number of lymphocytes (r = 0.56, p = 0.007). CONCLUSION: In COVID patients, thymus enlargement was frequent and associated with increased T lymphocyte production, which appears to be a beneficial adaptation to virus-induced lymphopaenia. The lack of thymic activity/reactivation in older SARS-CoV-2 infected patients could contribute to a worse prognosis.

TLR3-responsive, XCR1+, CD141(BDCA-3)+/CD8α+-equivalent dendritic cells uncovered in healthy and simian immunodeficiency virus-infected rhesus macaques.

In mice, CD8α(+) myeloid dendritic cells (mDC) optimally cross-present Ags to CD8(+) T cells and respond strongly to TLR3 ligands. Although equivalent DC have been identified by comparative genomic analysis and functional studies in humans as XCR1(+)CD141 (BDCA-3)(+)Clec9A(+)cell adhesion molecule 1(+) mDC, and in sheep as CD26(+) mDC, these cells remained elusive in nonhuman primates. To remedy this situation, we delineated precisely DC and monocyte populations by 12-color flow cytometry and transcriptomic analyses in healthy rhesus macaques. We identified a new mDC population, with strong phenotypic and transcriptional homology to human CD141(+) and murine CD8α(+) mDC, including XCR1 membrane expression as a conserved specific marker. In contrast, high CD11c expression was not characteristic of mDC in macaques, but of CD16(+) monocytes. Like their human and murine homologs, simian XCR1(+) mDC had much stronger responses to TLR3 stimulation than other myeloid cells. The importance of this new mDC population was tested in SIV(mac251) infection, the most relevant animal model for pathogenic HIV-1 infection and vaccination. This population increased sharply and transiently during acute infection, but was reduced in blood and spleen during advanced disease. The identification of XCR1(+) mDC in rhesus macaques opens new avenues for future preclinical vaccinal studies and highlights XCR1 as a prime candidate for targeted vaccine delivery.

Genetically determined thymic function affects strength and duration of immune response in COVID patients with pneumonia.

Thymic activation improves the outcome of COVID-19 patients with severe pneumonia. The rs2204985 genetic polymorphism within the TCRA-TCRD locus, which affects thymic output in healthy individuals, was found here to modify SARS-CoV-2-specific immunity and disease severity in COVID-19 patients with severe pneumonia. Forty patients with severe COVID-19 pneumonia were investigated. The GG genotype at the rs2204985 locus was associated, independently of age and sex, with stronger and long-lasting anti-SARS-CoV-2 helper and cytotoxic T cell responses 6 months after recovery. The GG genotype was also associated with less severe lung involvement, higher thymic production, and higher counts of blood naïve T lymphocytes, including recent thymic emigrants, and a larger population of activated stem cell memory CD4+ T cells. Overall, GG patients developed a more robust and sustained immunity to SARS-CoV-2. Polymorphism at rs2204985 locus should be considered as an additional predictive marker of anti-SARS-CoV-2 immune response.

IL-7-Adjuvanted Vaginal Vaccine Elicits Strong Mucosal Immune Responses in Non-Human Primates.

Mucosal immune responses are crucial in protecting against pathogens entering through mucosal surfaces. However, due to poor T-cell responsiveness upon mucosal antigenic stimulation, mucosal immunity remains difficult to obtain through vaccines and requires appropriate adjuvants. We previously demonstrated that administered systemically to healthy macaques or locally expressed in the intestinal mucosa of acutely SIV-infected macaques, interleukin-7 (IL-7) triggers chemokine expression and immune cell homing into mucosae, suggesting its important role in the development of mucosal immune responses. We therefore examined whether local delivery of recombinant glycosylated simian IL-7 (rs-IL-7gly) to the vaginal mucosa of rhesus macaques could prepare the lower female genital tract (FGT) for subsequent immunization and act as an efficient mucosal adjuvant. First, we showed that local administration of rs-IL-7gly triggers vaginal overexpression of chemokines and infiltration of mDCs, macrophages, NKs, B- and T-cells in the lamina propria while MamuLa-DR+ APCs accumulated in the epithelium. Subsequent mucosal anti-DT immunization in macaques resulted in a faster, stronger, and more persistent mucosal antibody response compared to DT-immunization alone. Indeed, we detected robust productions of DT-specific IgAs and IgGs in their vaginal secretions and identified cells secreting DT-specific IgAs in their vaginal mucosa and IgGs in draining lymph nodes. Finally, the expression of chemokines involved in the organization of tertiary lymphoid structures (TLS) was only increased in the vaginal mucosa of IL-7-adjuvanted immunized macaques. Interestingly, TLSs developed around PNAd+ high endothelial venules in their lower FGT sampled 2 weeks after the last immunization. Non-traumatic vaginal administration of rs-IL-7gly prepares the mucosa to respond to subsequent local immunization and allows the development of a strong mucosal immune response in macaques, through the chemokine-dependent recruitment of immune cells, the activation of mDCs and the formation of TLSs. The localization of DT-specific IgA+ plasma cells in the upper vaginal mucosa argues for their contribution to the production of specific immunoglobulins in the vaginal secretions. Our results highlight the potential of IL-7 as a potent mucosal adjuvant to stimulate the FGT immune system and elicit vaginal antibody responses to local immunization, which is the most promising way to confer protection against many sexually transmitted diseases.

HIV-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy.

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication.

HIV reservoir dynamics in HAART-treated poor immunological responder patients under IL-7 therapy.

OBJECTIVES: Recombinant Human IL-7 (rhIL-7) therapy allows reconstituting systemic and tissue-associated CD4 T-cell populations in HIV-infected poor immunological responder (PIR) patients. However, in-vitro studies suggest that the impact of rhIL-7 treatment on HIV-DNA loads in vivo remains questionable. DESIGN: We assessed the dynamics of circulating HIV-DNA loads in IL-7-treated HIV-infected PIR individuals. METHODS: Forty-one rhIL-7-treated and 16 control participants from the INSPIRE-3 clinical trial were included. Participants received three weekly subcutaneous injections of rhIL-7. HIV-DNA was quantified by nested quantitative PCR in white blood cells sampled at D0, D28 and M3 and expressed as per milliliters and per CD4 T-cell. Changes in HIV-DNA loads in the CD4 compartment at M3 were confirmed on sorted CD4 cells. RESULTS: Together with rhIL-7-induced T-cell expansion, we observed a significant raise in both infected cell frequencies and counts during the first 28 days of follow-up. During this period, HIV-DNA load per CD4 T-cell also increased, to a lower extent. Three months post-therapy, both the frequencies and counts of infected cells diminished in blood as compared with D28 but remained significantly higher than before IL-7 therapy. In contrast, infection frequencies strongly diminished within CD4 cells, reaching slightly but significantly lower levels than at baseline. CONCLUSION: rhIL-7 treatment initially drives an expansion of HIV reservoir in PIR patients by D28. This expansion is probably not only because of infected cell proliferation, but also to possible enhanced neoinfection, despite highly active antiretroviral therapy. In contrast, subsequent reduction in HIV-DNA load per CD4 T-cell argues for partial elimination of infected cells between D28 and M3.

Acute Simian Immunodeficiency Virus Infection Triggers Early and Transient Interleukin-7 Production in the Gut, Leading to Enhanced Local Chemokine Expression and Intestinal Immune Cell Homing.

The intestinal barrier, one of the first targets of HIV/simian immunodeficiency virus (SIV) is subjected to major physiological changes during acute infection. Having previously shown that pharmaceutical injection of interleukin-7 (IL-7) triggers chemokine expression in many organs leading to massive T-cell homing, in particular to the intestine, we here explored mucosal IL-7 expression as part of the cytokine storm occurring during the acute phase of SIV infection in rhesus macaques. Quantifying both mRNA and protein in tissues, we demonstrated a transient increase of IL-7 expression in the small intestine of SIV-infected rhesus macaques, starting with local detection of the virus by day 3 of infection. We also observed increased transcription levels of several chemokines in the small intestine. In infected macaques, ileal IL-7 expression correlated with the transcription of four of these chemokines. Among these chemokines, the macrophage and/or T-cell attractant chemokines CCL4, CCL25, and CCL28 also demonstrated increased transcription in uninfected IL-7-treated monkeys. Through immunohistofluorescence staining and image analysis, we observed increased CD8+ T-cell numbers and stable CD4+ T-cell counts in the infected lamina propria (LP) during hyperacute infection. Concomitantly, circulating CCR9+beta7+ CD4+ and CD8+ T-cells dropped during acute infection, suggesting augmented intestinal homing of gut-imprinted T-cells. Finally, CD4+ macrophages transiently decreased in the submucosa and concentrated in the LP during the first days of infection. Overall, our study identifies IL-7 as a danger signal in the small intestine of Chinese rhesus macaques in response to acute SIV infection. Through stimulation of local chemokine expressions, this overexpression of IL-7 triggers immune cell recruitment to the gut. These findings suggest a role for IL-7 in the initiation of early mucosal immune responses to SIV and HIV infections. However, IL-7 triggered CD4+ T-cells and macrophages localization at viral replication sites could also participate to viral spread and establishment of viral reservoirs.

Reversing Gut Damage in HIV Infection: Using Non-Human Primate Models to Instruct Clinical Research.

Antiretroviral therapy (ART) has led to dramatic improvements in the lives of HIV-infected persons. However, residual immune activation, which persists despite ART, is associated with increased risk of non-AIDS morbidities. Accumulating evidence shows that disruption of the gut mucosal epithelium during SIV/HIV infections allows translocation of microbial products into the circulation, triggering immune activation. This disruption is due to immune, structural and microbial alterations. In this review, we highlighted the key findings of gut mucosa studies of SIV-infected macaques and HIV-infected humans that have revealed virus-induced changes of intestinal CD4, CD8 T cells, innate lymphoid cells, myeloid cells, and of the local cytokine/chemokine network in addition to epithelial injuries. We review the interplay between the host immune response and the intestinal microbiota, which also impacts disease progression. Collectively, these studies have instructed clinical research on early ART initiation, modifiers of microbiota composition, and recombinant cytokines for restoring gut barrier integrity.

Modified interferon-α subtypes production and chemokine networks in the thymus during acute simian immunodeficiency virus infection, impact on thymopoiesis.

OBJECTIVES: Thymus dysfunction characterizes human/simian immunodeficiency virus (SIV) infections and contributes to physiopathology. However, both the mechanisms involved in thymic dysfunction and its precise timing remain unknown. We here analyzed thymic function during acute SIV infection in rhesus macaques. DESIGN AND METHODS: Rhesus macaques were intravenously infected with SIVmac251 and bled every 2/3 days or necropsied at different early time points postinfection. Naive T-cell counts were followed by flow cytometry and their T-cell receptor excision circle content evaluated by qPCR. Thymic chemokines were quantified by reverse transcription-qPCR and localized by in-situ hybridization in thymuses collected at necropsy. Thymic interferon alpha (IFN-α) subtype production was quantified by reverse transcription-qPCR combined to heteroduplex tracking assay. The effect of thymic IFN-α subtypes was tested on sorted triple negative thymocytes cultured on OP9-hDL1 cells. RESULTS: A reduced intrathymic proliferation history characterizes T cells produced during the first weeks of infection. Moreover, we evidenced a profound alteration of both chemokines and IFN-α subtypes transcriptional patterns in SIV-infected thymuses. Finally, we showed that IFN-α subtypes produced in the infected thymuses inhibit thymocyte proliferation, still preserving their differentiation capacity. CONCLUSION: Thymopoiesis is deeply impacted from the first days of SIV infection. Reduced thymocyte proliferation - a time-consuming process - together with modified chemokine networks is consistent with thymocyte differentiation speed-up. This may transiently enhance thymic output, thus increasing naive T-cell counts and diversity and the immune competence of the host. Nonetheless, long-lasting modification of thymic physiology may lead to thymic exhaustion, as observed in late primary HIV infection.

Chemokines at mucosal barriers and their impact on HIV infection.

Aside from representing a physical barrier and providing an unfavorable chemical milieu to viral and bacterial infections, mucosae of gut and female genital tract also contain organized lymphoid structures that support the initiation of anti-microbial immune responses, and more diffuse lymphoid tissues that represent immune effector mucosal sites. Local expression of specific chemokines orchestrates lymphoid cell trafficking and positioning in the mucosa-associated lymphoid tissues, leading to their efficient priming during antigenic stimulations as well as their specific homing back where they were primed. This review examines productions and roles of mucosae-specific chemokines in healthy and pathological conditions, as well as their possible positive and deleterious effects during mucosal HIV infection.

Rapid dissemination of SIV follows multisite entry after rectal inoculation.

Receptive ano-rectal intercourse is a major cause of HIV infection in men having sex with men and in heterosexuals. Current knowledge of the mechanisms of entry and dissemination during HIV rectal transmission is scarce and does not allow the development of preventive strategies. We investigated the early steps of rectal infection in rhesus macaques inoculated with the pathogenic isolate SIVmac251 and necropsied four hours to nine days later. All macaques were positive for SIV. Control macaques inoculated with heat-inactivated virus were consistently negative for SIV. SIV DNA was detected in the rectum as early as four hours post infection by nested PCR for gag in many laser-microdissected samples of lymphoid aggregates and lamina propria but never in follicle-associated epithelium. Scarce SIV antigen positive cells were observed by immunohistofluorescence in the rectum, among intraepithelial and lamina propria cells as well as in clusters in lymphoid aggregates, four hours post infection and onwards. These cells were T cells and non-T cells that were not epithelial cells, CD68(+) macrophages, DC-SIGN(+) cells or fascin(+) dendritic cells. DC-SIGN(+) cells carried infectious virus. Detection of Env singly spliced mRNA in the mucosa by nested RT-PCR indicated ongoing viral replication. Strikingly, four hours post infection colic lymph nodes were also infected in all macaques as either SIV DNA or infectious virus was recovered. Rapid SIV entry and dissemination is consistent with trans-epithelial transport. Virions appear to cross the follicle-associated epithelium, and also the digestive epithelium. Viral replication could however be more efficient in lymphoid aggregates. The initial sequence of events differs from both vaginal and oral infections, which implies that prevention strategies for rectal transmission will have to be specific. Microbicides will need to protect both digestive and follicle-associated epithelia. Vaccines will need to induce immunity in lymph nodes as well as in the rectum.

A role for the Rab6A' GTPase in the inactivation of the Mad2-spindle checkpoint.

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A', regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A' function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150(Glued), a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A' likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A', through its interaction with p150(Glued) and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.

Delayed viral replication and CD4(+) T cell depletion in the rectosigmoid mucosa of macaques during primary rectal SIV infection.

Rectal infection of macaques by SIV is a model for rectal HIV transmission. We focus here on the digestive tract during days 7-14 of primary rectal infection by SIV in 15 rhesus macaques. Surprisingly, we did not detect productively infected cells in the rectosigmoid colon at early stages of viral dissemination. This strongly suggests that there is no massive viral amplification in the rectosigmoid colon prior to viral dissemination. As dissemination proceeds, productively infected T cells are observed in the rectosigmoid colon and small intestine, with rectosigmoid colon showing the heaviest viral load. Lymphoid follicles are infected prior to lamina propria at both sites. When viral dissemination is widespread, inflammatory infiltrates are visible in the rectosigmoid colon, but not in the small intestine. An important decrease in CD4(+) T cells is then observed in the lamina propria of the rectosigmoid colon only.