Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Antiviral innate immunity is diminished in the upper respiratory tract of severe COVID-19 patients.

Understanding early innate immune responses to coronavirus disease 2019 (COVID-19) is crucial to developing targeted therapies to mitigate disease severity. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection elicits interferon expression leading to transcription of IFN-stimulated genes (ISGs) to control viral replication and spread. SARS-CoV-2 infection also elicits NF-κB signaling which regulates inflammatory cytokine expression contributing to viral control and likely disease severity. Few studies have simultaneously characterized these two components of innate immunity to COVID-19. We designed a study to characterize the expression of interferon alpha-2 (IFNA2) and interferon beta-1 (IFNB1), both type-1 interferons (IFN-1), interferon-gamma (IFNG), a type-2 interferon (IFN-2), ISGs, and NF-κB response genes in the upper respiratory tract (URT) of patients with mild (outpatient) versus severe (hospitalized) COVID-19. Further, we characterized the weekly dynamics of these responses in the upper and lower respiratory tracts (LRTs) and blood of severe patients to evaluate for compartmental differences. We observed significantly increased ISG and NF-κB responses in the URT of mild compared with severe patients early during illness. This pattern was associated with increased IFNA2 and IFNG expression in the URT of mild patients, a trend toward increased IFNB1-expression and significantly increased STING/IRF3/cGAS expression in the URT of severe patients. Our by-week across-compartment analysis in severe patients revealed significantly higher ISG responses in the blood compared with the URT and LRT of these patients during the first week of illness, despite significantly lower expression of IFNA2, IFNB1, and IFNG in blood. NF-κB responses, however, were significantly elevated in the LRT compared with the URT and blood of severe patients during peak illness (week 2). Our data support that severe COVID-19 is associated with impaired interferon signaling in the URT during early illness and robust pro-inflammatory responses in the LRT during peak illness.

Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19.

Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy.

Novel Majeed Syndrome-Causing LPIN2 Mutations Link Bone Inflammation to Inflammatory M2 Macrophages and Accelerated Osteoclastogenesis.

OBJECTIVE: To identify novel heterozygous LPIN2 mutations in a patient with Majeed syndrome and characterize the pathomechanisms that lead to the development of sterile osteomyelitis. METHODS: Targeted genetic analysis and functional studies assessing monocyte responses, macrophage differentiation, and osteoclastogenesis were conducted to compare the pathogenesis of Majeed syndrome to interleukin-1 (IL-1)-mediated diseases including neonatal-onset multisystem inflammatory disease (NOMID) and deficiency of the IL-1 receptor antagonist (DIRA). RESULTS: A 4-year-old girl of mixed ethnic background presented with sterile osteomyelitis and elevated acute-phase reactants. She had a 17.8-kb deletion on the maternal LPIN2 allele and a splice site mutation, p.R517H, that variably spliced out exons 10 and 11 on the paternal LPIN2 allele. The patient achieved long-lasting remission receiving IL-1 blockade with canakinumab. Compared to controls, monocytes and monocyte-derived M1-like macrophages from the patient with Majeed syndrome and those with NOMID or DIRA had elevated caspase 1 activity and IL-1ß secretion. In contrast, lipopolysaccharide-stimulated, monocyte-derived, M2-like macrophages from the patient with Majeed syndrome released higher levels of osteoclastogenic mediators (IL-8, IL-6, tumor necrosis factor, CCL2, macrophage inflammatory protein 1α/ß, CXCL8, and CXCL1) compared to NOMID patients and healthy controls. Accelerated osteoclastogenesis in the patient with Majeed syndrome was associated with higher NFATc1 levels, enhanced JNK/MAPK, and reduced Src kinase activation, and partially responded to JNK inhibition and IL-1 (but not IL-6) blockade. CONCLUSION: We report 2 novel compound heterozygous disease-causing mutations in LPIN2 in an American patient with Majeed syndrome. LPIN2 deficiency drives differentiation of proinflammatory M2-like macrophages and enhances intrinsic osteoclastogenesis. This provides a model for the pathogenesis of sterile osteomyelitis which differentiates Majeed syndrome from other IL-1-mediated autoinflammatory diseases.

Mucosal-associated invariant T (MAIT) cells provide B-cell help in vaccinated and subsequently SIV-infected Rhesus Macaques.

Mucosal-associated invariant T (MAIT) cells help combat opportunistic infections. Thus, MAIT cells are of interest in HIV/SIV vaccination and infection. We investigated MAIT cell dynamics and function in rhesus macaque blood and bronchoalveolar lavage (BAL) following mucosal adenovirus (Ad)-SIV recombinant priming, intramuscular SIV envelope boosting and infection following repeated low-dose intravaginal SIV exposures. Increased frequencies of blood MAIT cells over the course of vaccination were observed, which were maintained even 12-weeks post-SIV infection. BAL MAIT cells only increased after the first Ad immunization. Vaccination increased MAIT cell levels in blood and BAL expressing the antiviral cytokine IFN-γ and TNF-α and the proliferation marker Ki67. Upon T cell-specific α-CD3, α-CD28 stimulation, MAIT cells showed a greater capacity to secrete cytokines/chemokines associated with help for B cell activation, migration and regulation compared to CD3+MR1- cells. Culture of MAIT cell supernatants with B cells led to greater tissue like memory B cell frequencies. MAIT cell frequencies in blood and BAL correlated with SIV-specific antibody levels in rectal secretions and with SIV-specific tissue resident memory B cells. Overall, SIV vaccination influenced MAIT cell frequency and functionality. The potential for MAIT cells to provide help to B cells was evident during both vaccination and infection.

Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages.

Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.

Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages.

Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.

Fibrocytes Differ from Macrophages but Can Be Infected with HIV-1.

Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1-induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.

M-CSF inhibits anti-HIV-1 activity of IL-32, but they enhance M2-like phenotypes of macrophages.

M-CSF promotes the differentiation and survival of macrophages, and preferentially induces anti-inflammatory M2, rather than proinflammatory M1 macrophages. Recently, another cytokine, IL-32, was also shown to promote macrophage differentiation. In this article, we provide the first evidence, to our knowledge, that M-CSF has both additive and inhibitory effects on the macrophage-related activities of IL-32. When added to M-CSF-derived macrophages, M-CSF and IL-32 promoted macrophage survival, which was further enhanced by their combination. However, they had different effects on HIV-1 replication; that is, it was stimulated by M-CSF and inhibited by IL-32. Interestingly, the anti-HIV-1 activity of IL-32 was counteracted by M-CSF. Such inhibitory effect of M-CSF was not observed with IL-32-induced M1-like features including high cytokine/chemokine production and strong expression of the costimulatory molecule CD80. However, IL-32-treated macrophages unexpectedly showed also M2-like features including increased phagocytic activity, and high expression of CD14 and the scavenger receptor CD163, and the expression of CD14 and CD163 was further upregulated by cotreatment with M-CSF. The findings of this study regarding the unique functional interplay between M-CSF and IL-32 increase our understanding of the mechanisms that regulate the survival and M1/M2 ratio of macrophages, as well as HIV-1 replication in macrophages.

M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression.

The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.