T-Cell Activation Independently Associates With Immune Senescence in HIV-Infected Recipients of Long-term Antiretroviral Treatment.

BACKGROUND: Aging-associated noncommunicable comorbidities are more prevalent among human immunodeficiency virus type 1 (HIV)-infected individuals than among HIV-uninfected individuals. Residual HIV-related chronic immune activation and senescence may increase the risk of developing comorbidities. METHODS: Immune phenotyping, thymic output, and telomere length were assessed in 94 HIV-infected individuals who were aged >45 years and receiving antiretroviral therapy (ART; cases) and 95 age-matched uninfected controls. RESULTS: Cases had lower CD4(+) T-cell counts, higher CD8(+) T-cell counts, and increased levels of immune activation (ie, increased soluble CD14 [sCD14] level and increased percentages of CD38(+)HLA-DR(+) cells among both CD4(+) and CD8(+) T cells), regulatory T cells, and percentage of programmed cell death 1 (PD-1)-expressing cells among CD4(+) T cells. Immune senescence levels (ie, percentages of CD27(-)CD28(-) cells or CD57(+) cells) were comparable between cases and controls. Peripheral blood mononuclear cells from cases had shorter telomeres but increased single-joint T-cell receptor excision circle content and CD31(+) naive CD4(+) T cells. Although cytomegalovirus (CMV) antibody titers were higher in cases, CMV-specific T-cell responses were comparable between cases and controls. T-cell senescence in cases was independently associated with T-cell activation but not with CMV-specific immune responses. CONCLUSIONS: Despite long-term receipt of ART, HIV-infected adults had higher levels of immune activation, regulatory T cells, and PD-1-expressing CD4(+) cells and shorter telomeres. The increased soluble CD14 levels and percentage of CD38(+)HLA-DR(+) cells among CD4(+) T cells correlated with shorter telomeres and increased regulatory T-cell levels. This suggests that HIV influences immune function irreversibly, with several pathways that are persistently abnormal during effective ART. Therapies aimed at improving immune health during ART are needed.

Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4­activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.

Next-generation sequencing of microRNAs uncovers expression signatures in polarized macrophages.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at a posttranscriptional level and play a crucial role in the development of cells of the immune system. Macrophages are essential for generating inflammatory reactions upon tissue damage and encountering of invading pathogens, yet modulation of their immune responses is critical for maintaining tissue homeostasis. Macrophages can present different phenotypes, depending on the cytokine environment they encounter in the affected tissues. In this study, we have identified expression signatures of miRNAs that are differentially regulated during maturation of monocytes and polarization of macrophages by cytokines. We present a comprehensive characterization of miRNA expression in human monocytes and M1, M2a, and M2c polarized macrophages, using next-generation sequencing. Furthermore, we show that miRNA expression signatures are closely related to the various immune functions of polarized macrophages and therefore are involved in shaping the diverse phenotypes of these cells. The miRNAs identified here serve as markers for identification of inflammatory macrophages involved in the development of immune responses. Our findings contribute to understanding the role of miRNAs in determining the macrophage function in healthy and diseased tissues.

Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway.

Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.

Systematic review and meta-analysis of the factors affecting waning of post-vaccination neutralizing antibody responses against SARS-CoV-2.

Mass COVID-19 vaccination and continued introduction of new SARS-CoV-2 variants increased prevalence of hybrid immunity at various stages of waning protection. We systematically reviewed waning of post-vaccination neutralizing antibody titers in different immunological settings to investigate differences. We searched published and pre-print studies providing post-vaccination neutralizing antibody responses against the Index strain or Omicron BA.1. We used random effects meta-regression to estimate fold-reduction from months 1 to 6 post last dose by primary vs booster regimen and infection-naïve vs hybrid-immune cohorts. Among 26 eligible studies, 65 cohorts (range 3-21 per stratum) were identified. Month-1 titers varied widely across studies within each cohort and by vaccine platform, number of doses and number of prior infections. In infection-naïve cohorts, the Index strain waned 5.1-fold (95%CI: 3.4-7.8; n = 19 cohorts) post-primary regimen and 3.8-fold (95%CI: 2.4-5.9; n = 21) post-booster from months 1 to 6, and against Omicron BA.1 waned 5.9-fold (95%CI: 3.8-9.0; n = 16) post-booster; Omicron BA.1 titers post-primary were too low to assess. In hybrid-immune, post-primary cohorts, titers waned 3.7-fold (95%CI: 1.7-7.9; n = 8) against the Index strain and 5.0-fold (95%CI: 1.1-21.8; n = 6) against Omicron BA.1; post-booster studies of hybrid-immune cohorts were too few (n = 3 cohorts each strain) to assess. Waning was similar across vaccination regimen and prior-infection status strata but was faster for Omicron BA.1 than Index strains, therefore, more recent sub-variants should be monitored. Wide differences in peak titers by vaccine platform and prior infection status mean titers drop to non-protective levels sooner in some instances, which may affect policy.

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo.

Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

Creation of a nonspreading Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.

Post-vaccination T cell immunity to omicron.

In late 2021, the omicron variant of SARS Coronavirus 2 (SARS-CoV-2) emerged and replaced the previously dominant delta strain. Effectiveness of COVID-19 vaccines against omicron has been challenging to estimate in clinical studies or is not available for all vaccines or populations of interest. T cell function can be predictive of vaccine longevity and effectiveness against disease, likely in a more robust way than antibody neutralization. In this mini review, we summarize the evidence on T cell immunity against omicron including effects of boosters, homologous versus heterologous regimens, hybrid immunity, memory responses and vaccine product. Overall, T cell reactivity in post-vaccine specimens is largely preserved against omicron, indicating that vaccines utilizing the parental antigen continue to be protective against disease caused by the omicron variant.

CD9 and ITGA3 are regulated during HIV-1 infection in macrophages to support viral replication.

Monocytes/macrophages are important target cells for HIV-1. Here, we investigated whether HIV-1 induces changes in the macrophage gene expression profile to support viral replication. We observed that the macrophage gene expression profiles dramatically changed upon HIV-1 infection. The majority of the HIV-1 regulated genes were also differentially expressed in M2a macrophages. The biological functions associated with the HIV-1 induced gene expression profile in macrophages were mainly related to inflammatory responses. CD9 and ITGA3 were among the top genes upregulated upon HIV-1 infection. We showed that these genes support viral replication and that downregulation of these genes decreased HIV-1 replication in macrophages. Here we showed that HIV-1 infection of macrophages induces a gene expression profile that may dampen inflammatory responses. CD9 and ITGA3 were among the top genes regulated by HIV-1 and were shown to support viral production most likely at the level of viral budding and release.

Immunization with Pneumocystis recombinant KEX1 induces robust and durable humoral responses in immunocompromised non-human primates.

Infection with the opportunistic fungal pathogen, Pneumocystis jirovecii causes life-threatening pneumonia in immunocompromised individuals. In addition to HIV-1 infected patients, individuals at risk of Pneumocystis infection include those receiving immunosuppressive therapies due to transplantation, cancer or autoimmune disease. Antibiotic treatment is not always successful, and it does not prevent obstructive lung disease after clearance of the pathogen. Therefore, it is essential to develop therapeutic alternatives that are more effective against PCP. We reported that Pneumocystis recombinant protein KEX1 induces protective immunity against the development of PCP in a non-human primate model of HIV-induced immunosuppression. In this study, we tested the immunogenicity KEX1 immunization of healthy rhesus macaques and the durability of these responses during drug-induced immunosuppression using tacrolimus (FK506) and methylprednisolone. We observed that vaccination with KEX1 prior to the start of the immunosuppressive regimen generated a robust and long-lasting antibody response that was maintained throughout the immunosuppressive treatment. Furthermore, boosting with KEX1 during immunosuppression induced recall of memory responses against recombinant KEX1. The durability of the anti-KEX1 response and the ability to induce a recall response during immunosuppressive therapy provide a proof-of-concept data supporting further investigation of the KEX1 as a prophylactic vaccine to prevent PCP in drug-induced immunosuppression. This approach provides fundamental knowledge for the elaboration of therapeutic and prophylactic alternatives for PCP in patients undergoing severe immunosuppressive therapy.

G3BP1 restricts HIV-1 replication in macrophages and T-cells by sequestering viral RNA.

HIV-1 exploits the cellular machinery for replication and therefore several interactions with cellular factors take place, some of which are yet unknown. We identified GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) as a cellular factor that restricts HIV-1, by analyzing transcriptome profiles of in vitro-cytokine-activated macrophages that are non-permissive to HIV-1 replication. Silencing of G3BP1 by RNA interference resulted in increased HIV-1 replication in primary T-cells and macrophages, but did not affect replication of other retroviruses. G3BP1 specifically interacted with HIV-1 RNA in the cytoplasm, suggesting that it sequesters viral transcripts, thus preventing translation or packaging. G3BP1 was highly expressed in resting naïve or memory T-cells from healthy donors and HIV-1 infected patients, but significantly lower in IL-2-activated T-cells. These results strongly suggest that G3BP1 captures HIV-1 RNA transcripts and thereby restricts mRNA translation, viral protein production and virus particle formation.

Phosphodiesterase 8a supports HIV-1 replication in macrophages at the level of reverse transcription.

BACKGROUND: HIV-1 infected macrophages play a key role in HIV-1 infection. Even during anti-retroviral treatment, macrophages keep producing virus due to suboptimal tissue penetration and reduced efficacy of antiretrovirals. It is therefore of major importance to understand which host factors are involved in HIV-1 replication in macrophages. Previously, we have shown that genetic polymorphisms in phosphodiesterase 8a (PDE8A) are strongly associated with HIV-1 replication in these cells. Here we analyzed the mechanism and regulation of PDE8A in HIV-1 replication in macrophages. RESULTS: PDE8A mRNA expression strongly increases upon differentiation of monocytes into macrophages, which corresponds to the increased susceptibility of mature macrophages to HIV-1. In parallel, expression of microRNA miR-145-5p, predicted to target PDE8A mRNA, strongly decreased. The interaction of miR-145-5p with the 3' UTR of PDE8A mRNA could be experimentally validated, suggesting that indeed miR-145-5p can regulate PDE8A expression levels. Knockdown of PDE8A in macrophages resulted in a decrease in total HIV-1 replication and proviral DNA levels. These observations confirm that PDE8A regulates HIV-1 replication in macrophages and that this effect is mediated through early steps in the viral replication cycle. CONCLUSIONS: PDE8A is highly expressed in macrophages, and its expression is regulated by miR-145-5p. Our findings strongly suggest that PDE8A supports HIV-1 replication in macrophages and that this effect is mediated at the level of reverse transcription.

Next-generation sequencing of microRNAs in primary human polarized macrophages.

Macrophages are important for mounting inflammatory responses to tissue damage or infection by invading pathogens, and therefore modulation of their cellular functions is essential for the success of the immune system as well as for maintaining tissue homeostasis. Small non-coding RNAs are important regulatory elements of gene expression and microRNAs are the most widely known to be fundamental for the proper development of cells of the immune system. Macrophages can exhibit different phenotypes, depending on the cytokine environment they encounter in the affected tissues. We have analyzed the microRNA expression profiles during maturation of human primary monocytes into macrophages and polarization by pro- or anti-inflammatory cytokines. Here we describe the analysis of next-generation sequencing data deposited in EMBL-EBI ArrayExpress under accession number E-MTAB-1969 and associated with the study published by Cobos Jiménez and collaborators in Physiological Genomics in 2014 (1). The data presented here contributes to our understanding of microRNA expression profiles in human monocytes and macrophages and will also serve as a resource for novel microRNAs and other small RNA species expressed in these cells.

Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF) activity.

HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF) chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF) proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF) should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.

Differential expression of HIV-1 interfering factors in monocyte-derived macrophages stimulated with polarizing cytokines or interferons.

HIV-1 replication in macrophages can be regulated by cytokines and infection is restricted in macrophages activated by type I interferons and polarizing cytokines. Here, we observed that the expression levels of the cellular factors Trim5α, CypA, APOBEC3G, SAMHD-1, Trim22, tetherin and TREX-1, and the anti-HIV miRNAs miR-28, miR-150, miR-223 and miR-382 was upregulated by IFN-α and IFN-ß in macrophages, which may account for the inhibiting effect on viral replication and the antiviral state of these cells. Expression of these factors was also increased by IFN-γ +/- TNF-α, albeit to a lesser extent; yet, HIV-1 replication in these cells was not restricted at the level of proviral synthesis, indicating that these cellular factors only partially contribute to the observed restriction. IL-4, IL-10 or IL-32 polarization did not affect the expression of cellular factors and miRNAs, suggesting only a limited role for these cellular factors in restricting HIV-1 replication in macrophages.

Macrophages and HIV-1.

PURPOSE OF REVIEW: Macrophages play an important role in HIV-1 pathogenesis and contribute to the establishment of the viral reservoir responsible for continuous virus production. This review will discuss new insights into HIV-1 infection in macrophages and the effect of infection on immune function and pathology. RECENT FINDINGS: New cellular factors interacting with various steps of the HIV-1 replication cycle, such as entry, integration, transcription, and assembly of new viral progeny, have been identified. Cellular and viral microRNAs have been shown to regulate virus replication, promote viral latency, and prolong cell survival. Interference with innate immune functions, like phagocytosis, autophagy, cytokine production, and T-cell activation by HIV-1 has been found to contribute to virus replication and latency. Growing evidence indicates an important role of infected macrophages in a variety of HIV-1-associated diseases, including neurocognitive disorders. SUMMARY: Under combined antiretroviral therapy (cART), HIV-1 continues to persist in macrophages. Better understanding of HIV-1 infection in macrophages may lead to new adjunctive therapies to improve cART, specifically targeting the viral reservoir and ameliorating tissue-specific diseases.