Despite the efficacy of SARS-CoV-2 vaccines in reducing mortality and severe cases of COVID-19, a proportion of survivors experience long-term symptoms, known as post-acute sequelae of SARS-CoV-2 infection (PASC). This study investigates the long-term immunological and neurodegenerative effects associated with extracellular vesicles (EVs) in COVID-19 survivors, 15 months after SARS-CoV-2 infection. 13 Controls and 20 COVID-19 survivors, 15 months after SARS-CoV-2 infection, were recruited. Pro-inflammatory cytokines were analyzed in both plasma and EVs. A deep-immunophenotyping of monocytes, T-cells and dendritic cells (DCs) was performed, along with immunostainings of SARS-CoV-2 in the colon. Higher concentrations of pro-inflammatory cytokines and neurofilaments were found in EVs but not in plasma from COVID-19 survivors. Additionally, COVID-19 participants exhibited altered monocyte activation markers and elevated cytokine production upon lipopolysaccharide stimulation. Increased activation markers in CD4+ T-cells and decreased indoleamine 2,3-dioxygenase expression in DCs were observed in COVID-19 participants. Furthermore, the amount of plasmacytoid DCs expressing β7-integrin were higher in COVID-19, potentially associated with the viral persistence observed in the colon. COVID-19 survivors exhibit long-term immune dysregulation and neurodegeneration, emphasizing the need for ongoing monitoring of PASC. The cargo of EVs can be a promising tool for early detection of virus-induced neurological disorders.

Dendritic cells (DC) are known to modulate antiviral immune responses; however, the knowledge about the role of different DC subsets in antiviral T cell priming in human tissues remains uncompleted. In the context of HIV infection, we determined the phenotype and location of plasmacytoid and CD141+ myeloid DCs (pDCs and mDCs) in lymph nodes of people living with HIV (PLWH). We found an interaction between pDCs and CD141+ mDCs with CD8+ T cells, being associated with participants' viral levels in blood and tissue. Moreover, we demonstrated a higher and more polyfunctional superantigen- and HIV-specific CD8+ T cell response after the coculture with Toll-like receptor (TLR)-primed pDCs and CD141+ mDCs. Last, we showed the potential of programmed cell death-1 (PD-1) blocking using pembrolizumab to further increase antigen-specific CD8+ T cell response along with TLR agonists. Therefore, these results showed a cooperation between pDCs, CD141+ mDCs and CD8+ T cells in lymph nodes of PLWH, which is associated with higher HIV control, highlighting the importance of DC subsets crosstalk to achieve a more potent anti-HIV response and support the use of DC-based immunotherapies for HIV control.

The ubiquitin proteasome system (UPS) is implicated in protein homeostasis. One of the proteins involved in this system is HERC1 E3 ubiquitin ligase, which was associated with several processes including the normal development and neurotransmission at the neuromuscular junction (NMJ), autophagy in projection neurons, myelination of the peripheral nervous system, among others. The tambaleante (tbl) mouse model carries the spontaneous mutation Gly483Glu substitution in the HERC1 E3 protein. Using this model, we analyzed the implication of HERC1 E3 ubiquitin ligase in the activity of UPS, autophagy, and synaptic homeostasis in brain and muscle tissues. Regarding UPS, no differences were found in its activity nor in the specific gene expression in both brain and muscle tissues from tbl compared with the control littermates. Furthermore, the use of the specific UPS inhibitor (MG-132), did not alter the evoked neurotransmitter release in the levator auris longus (LAL) muscle. Interestingly, the expression of the autophagy-related gene p62 was significantly increased in the muscle of tbl compared to the control littermates. Indeed, impaired evoked neurotransmitter release was observed with the autophagy inhibitor Wortmannin. Finally, altered levels of Clathrin and Synaptophysin were detected in muscle tissues. Altogether, our findings show that HERC1 E3 ubiquitin ligase mutation found in tbl mice alters autophagy and vesicular recycling without affecting proteasomal function.

BACKGROUND We evaluated the safety and viral rebound, after analytical treatment interruption (ATI), of vedolizumab and ART in recent HIV-1 infection. We used this model to analyze the effect of α4β7 on the HIV-1 reservoir size.METHODS Participants started ART with monthly vedolizumab infusions, and ATI was performed at week 24. Biopsies were obtained from ileum and cecum at baseline and week 24. Vedolizumab levels, HIV-1 reservoir, flow cytometry, and cell-sorting and antibody competition experiments were assayed.RESULTS Vedolizumab was safe and well tolerated. No participant achieved undetectable viremia off ART 24 weeks after ATI. Only a modest effect on the time to achieve more than 1,000 HIV-1 RNA copies/mL and the proportion of participants off ART was observed, being higher in the vedolizumab group compared with historical controls. Just before ATI, α4β7 expression was associated with HIV-1 DNA and RNA in peripheral blood and with PD1 and TIGIT levels. Importantly, a complete blocking of α4β7 was observed on peripheral CD4+ T cells but not in gut (ileum and cecum), where α4β7 blockade and vedolizumab levels were inversely associated with HIV-1 DNA.CONCLUSION Our findings support α4β7 as an important determinant in HIV-1 reservoir size, suggesting the complete α4β7 blockade in tissue as a promising tool for HIV-cure combination strategies.TRIAL REGISTRATION ClinicalTrials.gov NCT03577782.FUNDING This work was supported by the Instituto de Salud Carlos III (Fondo Europeo de Desarrollo Regional, “a way to make Europe,” research contracts FI17/00186 and FI19/00083 and research projects PI18/01532, PI19/01127, PI22/01796), Conserjería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (research projects P20/00906), the Red Temática de Investigación Cooperativa en SIDA (RD16/0025/0020), and the Spanish National Research Council.

Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc). Apoptosis is thought to play a critical role in the progression of PD, and thus understanding the effects of antiapoptotic strategies is crucial for developing potential therapies. In this study, we developed a unique genetic model to selectively delete Casp3, the gene encoding the apoptotic protein caspase-3, in dopaminergic neurons (TH-C3KO) and investigated its effects in response to a subacute regime of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, which is known to trigger apoptotic loss of SNpc dopaminergic neurons. We found that Casp3 deletion did not protect the dopaminergic system in the long term. Instead, we observed a switch in the cell death pathway from apoptosis in wild-type mice to necrosis in TH-C3KO mice. Notably, we did not find any evidence of necroptosis in our model or in in vitro experiments using primary dopaminergic cultures exposed to 1-methyl-4-phenylpyridinium in the presence of pan-caspase/caspase-8 inhibitors. Furthermore, we detected an exacerbated microglial response in the ventral mesencephalon of TH-C3KO mice in response to MPTP, which mimicked the microglia neurodegenerative phenotype (MGnD). Under these conditions, it was evident the presence of numerous microglial phagocytic cups wrapping around apparently viable dopaminergic cell bodies that were inherently associated with galectin-3 expression. We provide evidence that microglia exhibit phagocytic activity towards both dead and stressed viable dopaminergic neurons through a galectin-3-dependent mechanism. Overall, our findings suggest that inhibiting apoptosis is not a beneficial strategy for treating PD. Instead, targeting galectin-3 and modulating microglial response may be more promising approaches for slowing PD progression.

Early infection in life has been implicated in increasing the risk for neurological disorders. Here we performed single-cell sequencing of microglia and monocytes from 6-month-old WT and 5xFAD mice subjected to one dose of LPS (1mg/kg) at postnatal day 9. We successfully mapped disease-associated microglia (DAM) and perivascular macrophages in our data and demonstrated a subpopulation of microglia that adopted a monocyte-like profile, marked by Lyz2, Tmsb10, Lgals1and Lgals3. This unique subset appeared in response to early systemic LPS challenge and AD pathology but diminished in the presence of double stimulus. Different cytokines were altered in the brain and periphery as seen using mesoscale plates. GM-CSF and MIP-1beta levels were altered in an amyloid-beta(A beta)-dependent manner in hippocampus. MIP-1beta and IFN-gamma were altered upon early LPS stimulation. In the periphery, we found MMP-9 was significantly increased in serum samples from 5xFAD mice. Interestingly, early LPS stimulation significantly elevated TNF-alpha in serum from WT and 5xFAD mice, but was reduced in the hippocampus due to A beta pathology. The LPS treatment in 5xFAD mice had a tendency to improve the short-term memory deficit. Taken together, we observed long-lasting effects from early life stress, including activation of inflammation in the periphery and brain through modulation of different signaling cascades. ### Competing Interest Statement The authors have declared no competing interest.

The HIV attacks the immune system provoking an infection that is considered a global health challenge. Despite antiretroviral treatments being effective in reducing the plasma viral load in the blood to undetectable levels in people living with HIV (PLWH), the disease is not cured and has become chronic. This happens because of the existence of anatomical and cellular viral reservoirs, mainly located in the lymph nodes and gastrointestinal tract, which are composed of infected CD4+ T cells with a resting memory phenotype and inaccessible to antiretroviral therapy. Herein, a new therapeutic strategy based on nanotechnology is presented. Different combinations of antiretroviral drugs (bictegravir/tenofovir/emtricitabine and nevirapine/tenofovir/emtricitabine) and toll-like receptor agonists were encapsulated into metal-organic frameworks (MOFs) PCN-224 and ZIF-8. The encapsulation efficiencies of all the drugs, as well as their release rate from the carriers, were measured. In vitro studies about the cell viability, the hemocompatibility, and the platelet aggregation of the MOFs were carried out. Epifluorescence microscopy assays confirmed the ability of ZIF-8 to target a carboxyfluorescein probe inside HeLa cell lines and PBMCs. These results pave the way for the use of these structures to eliminate latent HIV reservoirs from anatomical compartments through the activation of innate immune cells, and a higher efficacy of the triplet combinations of antiretroviral drugs.

BACKGROUNDPersistent controllers (PCs) maintain antiretroviral-free HIV-1 control indefinitely over time, while transient controllers (TCs) eventually lose virological control. It is essential to characterize the quality of the HIV reservoir in terms of these phenotypes in order to identify the factors that lead to HIV progression and to open new avenues toward an HIV cure.METHODSThe characterization of HIV-1 reservoir from peripheral blood mononuclear cells was performed using next-generation sequencing techniques, such as full-length individual and matched integration site proviral sequencing (FLIP-Seq; MIP-Seq).RESULTSPCs and TCs, before losing virological control, presented significantly lower total, intact, and defective proviruses compared with those of participants on antiretroviral therapy (ART). No differences were found in total and defective proviruses between PCs and TCs. However, intact provirus levels were lower in PCs compared with TCs; indeed the intact/defective HIV-DNA ratio was significantly higher in TCs. Clonally expanded intact proviruses were found only in PCs and located in centromeric satellite DNA or zinc-finger genes, both associated with heterochromatin features. In contrast, sampled intact proviruses were located in permissive genic euchromatic positions in TCs.CONCLUSIONSThese results suggest the need for, and can give guidance to, the design of future research to identify a distinct proviral landscape that may be associated with the persistent control of HIV-1 without ART.FUNDINGInstituto de Salud Carlos III (FI17/00186, FI19/00083, MV20/00057, PI18/01532, PI19/01127 and PI22/01796), Gilead Fellowships (GLD22/00147). NIH grants AI155171, AI116228, AI078799, HL134539, DA047034, MH134823, amfAR ARCHE and the Bill and Melinda Gates Foundation.

Adult hippocampal neurogenesis (AHN) is a process involved in numerous neurodegenerative diseases. Many researchers have described microglia as a key component in regulating the formation and migration of new neurons along the rostral migratory stream. Caspase-3 is a cysteine-aspartate-protease classically considered as one of the main effector caspases in the cell death program process. In addition to this classical function, we have identified the role of this protein as a modulator of microglial function; however, its action on neurogenic processes is unknown. The aim of the present study is to identify the role of Caspase-3 in neurogenesis-related microglial functions. To address this study, Caspase-3 conditional knockout mice in the microglia cell line were used. Using this tool, we wanted to elucidate the role of this protein in microglial function in the hippocampus, the main region in which adult neurogenesis takes place. After the reduction of Caspase-3 in microglia, mutant mice showed a reduction of microglia in the hippocampus, especially in the dentate gyrus region, a region inherently associated to neurogenesis. In addition, we found a reduction in doublecortin-positive neurons in conditional Caspase-3 knockout mice, which corresponds to a reduction in neurogenic neurons. Furthermore, using high-resolution image analysis, we also observed a reduction in the phagocytic capacity of microglia lacking Caspase-3. Behavioral analysis using object recognition and Y-maze tests showed altered memory and learning in the absence of Caspase-3. Finally, we identified specific microglia located specifically in neurogenic niche positive for Galectin 3 which colocalized with Cleaved-Caspase-3 in control mice. Taken together, these results showed the essential role of Caspase-3 in microglial function and highlight the relevant role of this specific microglial phenotype in the maintenance of AHN in the hippocampus.

Microglia have an innate immunity memory (IIM) with divergent functions in different animal models of neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by chronic neuroinflammation, neurodegeneration, tau tangles and ss-amyloid (A ss) deposition. Systemic inflammation has been implicated in contributing to the progression of AD. Multiple reports have demonstrated unique microglial signatures in AD mouse models and patients. However, the proteomic profiles of microglia modified by IIM have not been well-documented in an AD model. Therefore, in the present study, we investigate whether lipopolysaccharide (LPS)-induced IIM in the pre-clinical stage of AD alters the microglial responses and shapes the neuropathology. We accomplished this by priming 5xFAD and wild-type (WT) mice with an LPS injection at 6 weeks (before the robust development of plaques). 140 days later, we evaluated microglial morphology, activation, the microglial barrier around A ss, and A ss deposition in both 5xFAD primed and unprimed mice. Priming induced decreased soma size of microglia and reduced colocalization of PSD95 and Synaptophysin in the retrosplenial cortex. Priming appeared to increase phagocytosis of A ss, resulting in fewer Thioflavin S+ A ss fibrils in the dentate gyrus. RIPA-soluble A ss 40 and 42 were significantly reduced in Primed-5xFAD mice leading to a smaller size of MOAB2(+) A ss plaques in the prefrontal cortex. We also found that A ss-associated microglia in the Primed-5xFAD mice were less activated and fewer in number. After priming, we also observed improved memory performance in 5xFAD. To further elucidate the molecular mechanism underlying these changes, we performed quantitative proteomic analysis of microglia and bone marrow monocytes. A specific pattern in the microglial proteome was revealed in primed 5xFAD mice. These results suggest that the imprint signatures of primed microglia display a distinctive phenotype and highlight

Background: Alzheimer’s disease (AD) is a progressive multifaceted neurodegenerative disorder for which no disease-modifying treatment exists. Neuroinflammation is central to the pathology progression, with evidence suggesting that microglia-released galectin 3 (gal3) plays a pivotal role by amplifying neuroinflammation in AD. However, possible involvement of gal3 in the disruption of cognition-relevant neuronal network oscillations typical of AD remains unknown. Methods: Here, we investigate the functional implications of gal3 signaling on cognition-relevant gamma oscillations (30-80 Hz) by performing electrophysiological recordings in hippocampal area CA3 of wild-type (WT) and 5xFAD mice in vitro . Results: Gal3 application decreases gamma oscillation power and rhythmicity in an activity-dependent manner and is accompanied by impairment of cellular dynamics in fast-spiking interneurons (FSN) and pyramidal cells (PCs). We found that gal3-induced disruption is mediated by the gal3-carbohydrate-recognition domain and prevented by the gal3 inhibitor TD139, which also prevents Aβ42-induced degradation of gamma oscillations. Furthermore, we demonstrate that 5xFAD mice lacking gal3 (5xFAD-Gal3KO) exhibit WT-like gamma network dynamics. Conclusions: We report for the first time that gal3 impairs cognition-relevant neuronal network dynamics by spike-phase uncoupling of FSN inducing a network performance collapse. Moreover, our findings suggest gal3 inhibition as a potential therapeutic target to counteract the neuronal network instability typical of AD and other neurological disorders encompassing neuroinflammation and cognitive decline.

BACKGROUND:Plasmacytoid dendritic cells (pDCs) sense viral and bacterial products through Toll-like receptor (TLR)-7 and -9 and translate this sensing into Interferon-α (IFN-α) production and T-cell activation. The understanding of the mechanisms involved in pDCs stimulation may contribute to HIV-cure immunotherapeutic strategies. The objective of the present study was to characterize the immunomodulatory effects of TLR agonist stimulations in several HIV-1 disease progression phenotypes and in non HIV-1 infected donors. METHODS:pDCs, CD4 and CD8 T-cells were isolated from 450 ml of whole blood from non HIV-1 infected donors, immune responders (IR), immune non responders (INR), viremic (VIR) and elite controller (EC) participants. pDCs were stimulated overnight with AT-2, CpG-A, CpG-C and GS-9620 or no stimuli. After that, pDCs were co-cultured with autologous CD4 or CD8 T-cells and with/without HIV-1 (Gag peptide pool) or SEB (Staphylococcal Enterotoxin B). Cytokine array, gene expression and deep immunophenotyping were assayed. FINDINGS:pDCs showed an increase of activation markers levels, interferon related genes, HIV-1 restriction factors and cytokines levels after TLR stimulation in the different HIV-disease progression phenotypes. This pDC activation was prominent with CpG-C and GS-9620 and induced an increase of HIV-specific T-cell response even in VIR and INR comparable with EC. This HIV-1 specific T-cell response was associated with the upregulation of HIV-1 restriction factors and IFN-α production by pDC. INTERPRETATION:These results shed light on the mechanisms associated with TLR-specific pDCs stimulation associated with the induction of a T-cell mediated antiviral response which is essential for HIV-1 eradication strategies. FUNDING:This work was supported by Gilead fellowship program, the Instituto de Salud Carlos III (Fondo Europeo de Desarrollo Regional, FEDER, "a way to make Europe") and the Red Temática de Investigación Cooperativa en SIDA and by the Spanish National Research Council (CSIC).

Alzheimer's disease (AD) is the most common neurodegenerative disease. Most cases of AD are considered idiopathic and likely due to a combination of genetic, environmental, and lifestyle-related risk factors. Despite occurring decades before the typical age of an AD diagnosis, early-life stress (ELS) has been suggested to have long-lasting effects that may contribute to AD risk and pathogenesis. Still, the mechanisms that underlie the role of ELS on AD risk remain largely unknown. Here, we used 5xFAD transgenic mice to study relatively short-term alterations related to ELS in an AD-like susceptible mouse model at 6 weeks of age. To model ELS, we separated pups from their dams for 3 h per day from postnatal day 2-14. Around 6 weeks of age, we found that maternally separated (MS) 5xFAD mice, particularly female mice, displayed increased amyloid-β-immunoreactivity in the anterior cingulate cortex (ACC) and basolateral amygdala (BLA). In anterior cingulate cortex, we also noted significantly increased intraneuronal amyloid-β-immunoreactivity associated with MS but only in female mice. Moreover, IBA1-positive DAPI density was significantly increased in relation to MS in ACC and BLA, and microglia in BLA of MS mice had significantly different morphology compared to microglia in non-MS 5xFAD mice. Cytokine analysis showed that male MS mice, specifically, had increased levels of neuroinflammatory markers CXCL1 and IL-10 in hippocampal extracts compared to non-MS counterparts. Additionally, hippocampal extracts from both male and female MS 5xFAD mice had decreased levels of synapse- and activity-related markers Bdnf, 5htr6, Cox2, and Syp in hippocampus. Lastly, we performed behavioral tests to evaluate anxiety- and depressive-like behavior and working memory but could not detect any significant differences between groups. Overall, we detected several sex-specific molecular and cellular alterations in 6-week-old adolescent 5xFAD mice associated with MS that may help explain the connection between ELS and AD risk.

Some HIV controllers experience immunologic progression with CD4(+) T cell decline. We aimed to identify genetic factors associated with CD4(+) T cell lost in HIV controllers. A total of 561 HIV controllers were included, 442 and 119 from the International HIV controllers Study Cohort and the Swiss HIV Cohort Study, respectively. No SNP or gene was associated with the long-term non-progressor HIV spontaneous control phenotype in the individual GWAS or in the meta-analysis. However, SNPs previously associated with natural HIV control linked to HLA-B (rs2395029 [p = 0.005; OR = 1.70], rs59440261 [p = 0.003; OR = 1.78]), MICA (rs112243036 [p = 0.011; OR = 1.45]), and PSORS1C1 loci (rs3815087 [p = 0.017; OR = 1.39]) showed nominal association with this phenotype. Genetic factors associated with the long-term HIV controllers without risk of immunologic progression are those previously related to the overall HIV controller phenotype.

SARS-CoV-2 specific T-cell response has been associated with disease severity, immune memory and heterologous response to endemic coronaviruses. However, an integrative approach combining a comprehensive analysis of the quality of SARS-CoV-2 specific T-cell response with antibody levels in these three scenarios is needed. In the present study, we found that, in acute infection, while mild disease was associated with high T-cell polyfunctionality biased to IL-2 production and inversely correlated with anti-S IgG levels, combinations only including IFN-γ with the absence of perforin production predominated in severe disease. Seven months after infection, both non-hospitalised and previously hospitalised patients presented robust anti-S IgG levels and SARS-CoV-2 specific T-cell response. In addition, only previously hospitalised patients showed a T-cell exhaustion profile. Finally, combinations including IL-2 in response to S protein of endemic coronaviruses were the ones associated with SARS-CoV-2 S-specific T-cell response in pre-COVID-19 healthy donors' samples. These results could have implications for protective immunity against SARS-CoV-2 and recurrent COVID-19 and may help for the design of new prototypes and boosting vaccine strategies.

The immune factors associated with impaired SARS-CoV-2 vaccine response in elderly people are mostly unknown. We studied individuals older than 60 and younger than 60 years, who had been vaccinated with SARS-CoV-2 BNT162b2 mRNA, before and after the first and second dose. Aging was associated with a lower anti-RBD IgG levels and a decreased magnitude and polyfunctionality of SARS-CoV-2-specific T cell response. The dramatic decrease in thymic function in people > 60 years, which fueled alteration in T cell homeostasis, and their lower CD161+ T cell levels were associated with decreased T cell response 2 months after vaccination. Additionally, deficient DC homing, activation, and TLR-mediated function, along with a proinflammatory functional profile in monocytes, were observed in the > 60-year-old group, which was also related to lower specific T cell response after vaccination. These findings might be relevant for the improvement of the current vaccination strategies and for the development of new vaccine prototypes.

Background The risk of developing Alzheimer’s disease (AD) is modulated by genetic and environmental factors. Early-life stress (ELS) exposure during critical periods of brain development can impact later brain function and health, including increasing the risk of developing AD. Microglial dysfunction and neuroinflammation have been implicated as playing a role in AD pathology and may be modulated by ELS. To complicate matters further, sex-specific effects have been noted in response to ELS and in the incidence and progression of AD. Methods Here, we subjected male and female mice with either a wild type or 5xFAD familial AD-model background to maternal separation (MS) from postnatal day 2 to 14 to induce ELS. Results We detected hippocampal neuroinflammatory alterations already at postnatal day 15. By 4 months of age, MS mice presented increased immobility time in the forced swim test and a lower discrimination index in the novel object recognition memory test compared to controls. We found altered Bdnf and Arc expression in the hippocampus and increased microglial activation in the prefrontal cortex due to MS in a sex-dependent manner. In 5xFAD mice specifically, MS exacerbated amyloid-beta deposition, particularly in females. In the periphery, the immune cell population was altered by MS exposure. Conclusion Overall, our results demonstrate that MS has both short- and long-term effects on brain regions related to memory and on the inflammatory system, both in the brain and periphery. These ELS-related effects that are detectable even in adulthood may exacerbate pathology and increase the risk of developing AD via sex-specific mechanisms.

SUMMARY The immune factors associated with impaired SARS-CoV-2 vaccine response in the elderly are mostly unknown. We studied old and young people vaccinated with SARS-CoV-2 BNT162b2 mRNA before and after the first and second dose. Aging was associated with a lower anti-RBD IgG levels and a decreased magnitude and polyfunctionality of SARS-CoV-2 specific T cell response. The dramatic decrease in thymic function in the elderly, which fueled alteration in T cell homeostasis, and lower CD161+ T cell levels were associated with decreased T cell response two months after vaccination. Additionally, a deficient dendritic cell (DC) homing, activation and Toll like receptor (TLR)-mediated function, along with a proinflammatory functional profile in monocytes, were observed in the elderly, which was also related to lower specific T cell response after vaccination. These findings might be relevant for the improvement of the current vaccination strategies and for the development of new vaccine prototypes.