ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Human rhinovirus 16 induces an ICAM-1-PKR-ATF2 axis to modulate macrophage functions.

Human rhinovirus (HRV) infections are the leading cause of disease exacerbations in individuals with chronic pulmonary diseases, primarily due to impaired macrophage functions, resulting in defective bacterial elimination. We previously demonstrated that HRV16 impairs macrophages' functions in an ARL5b-dependent manner. In permissive cells, ARL5b acted as an HRV16 restriction factor and was repressed. Here, we delve into the dual regulation of ARL5b by HRV16 in both cell types. We analyzed the effect of HRV16 on primary human macrophages using neutralizing antibodies, specific inhibitors, siRNA, and chromatin immune precipitation. Our study reveals that, while the virus does not replicate in macrophages, it induces interferon and pro-inflammatory responses. We identify the ICAM-1-PKR-ATF2 signaling axis as crucial for ARL5b induction in macrophages, whereas only ICAM-1 plays a role in ARL5b repression in permissive cells. Furthermore, HRV16 triggers epigenetic reprogramming in both cell types at the ARL5b promoter. In macrophages, epigenetic changes are ATF2 dependent. In conclusion, our findings highlight previously unknown signaling pathways activated by HRV16 in macrophages. Targeting these pathways could offer novel strategies to improve outcomes for individuals with respiratory conditions. IMPORTANCE: Human rhinovirus (HRV) infections are the leading cause of disease exacerbations in individuals with chronic pulmonary conditions and are frequently associated with bacterial superinfections due to defective bacterial elimination by macrophages. We previously identified ARL5b-induction by HRV16 to be responsible for the impairment of bacteria elimination. In contrast, in permissive cells, ARL5b is repressed and acts as a restriction factor for HRV16. Here, we investigated the dual regulation of ARL5b by HRV16 in these cells. Our study reveals that the ICAM-1-PKR-ATF2 signaling axis is crucial for ARL5b induction in macrophages. In permissive cells, only ICAM-1 plays a role in ARL5b repression. Moreover, HRV16 triggered epigenetic reprogramming in macrophages. ARL5b promoter was repressed in an ATF2-dependent manner. Collectively, our findings reveal previously unknown signaling pathways activated by HRV16 in macrophages. Targeting these pathways provides novel strategies to target ARL5b expression specifically in macrophages and improve outcomes for individuals with respiratory pathologies.

Zika virus infection of mature neurons from immunocompetent mice generates a disease-associated microglia and a tauopathy-like phenotype in link with a delayed interferon beta response.

BACKGROUND: Zika virus (ZIKV) infection at postnatal or adult age can lead to neurological disorders associated with cognitive defects. Yet, how mature neurons respond to ZIKV remains substantially unexplored. METHODS: The impact of ZIKV infection on mature neurons and microglia was analyzed at the molecular and cellular levels, in vitro using immunocompetent primary cultured neurons and microglia, and in vivo in the brain of adult immunocompetent mice following intracranial ZIKV inoculation. We have used C57BL/6 and the genetically diverse Collaborative Cross mouse strains, displaying a broad range of susceptibility to ZIKV infection, to question the correlation between the effects induced by ZIKV infection on neurons and microglia and the in vivo susceptibility to ZIKV. RESULTS: As a result of a delayed induction of interferon beta (IFNB) expression and response, infected neurons displayed an inability to stop ZIKV replication, a trait that was further increased in neurons from susceptible mice. Alongside with an enhanced expression of ZIKV RNA, we observed in vivo, in the brain of susceptible mice, an increased level of active Iba1-expressing microglial cells occasionally engulfing neurons and displaying a gene expression profile close to the molecular signature of disease-associated microglia (DAM). In vivo as well as in vitro, only neurons and not microglial cells were identified as infected, raising the question of the mechanisms underlying microglia activation following brain ZIKV infection. Treatment of primary cultured microglia with conditioned media from ZIKV-infected neurons demonstrated that type-I interferons (IFNs-I) secreted by neurons late after infection activate non-infected microglial cells. In addition, ZIKV infection induced pathological phosphorylation of Tau (pTau) protein, a hallmark of neurodegenerative tauopathies, in vitro and in vivo with clusters of neurons displaying pTau surrounded by active microglial cells. CONCLUSIONS: We show that ZIKV-infected mature neurons display an inability to stop viral replication in link with a delayed IFNB expression and response, while signaling microglia for activation through IFNs-I secreted at late times post-infection. In the brain of ZIKV-infected susceptible mice, uninfected microglial cells adopt an active morphology and a DAM expression profile, surrounding and sometimes engulfing neurons while ZIKV-infected neurons accumulate pTau, overall reflecting a tauopathy-like phenotype.

Arpin is critical for phagocytosis in macrophages and is targeted by human rhinovirus.

Human rhinovirus is a causative agent of severe exacerbations of chronic obstructive pulmonary disease (COPD). COPD is characterised by an increased number of alveolar macrophages with diminished phagocytic functions, but how rhinovirus infection affects macrophage function is still unknown. Here, we describe that human rhinovirus 16 impairs bacterial uptake and receptor-mediated phagocytosis in macrophages. The stalled phagocytic cups contain accumulated F-actin. Interestingly, we find that human rhinovirus 16 downregulates the expression of Arpin, a negative regulator of the Arp2/3 complex. Importantly, re-expression of the protein rescues defective internalisation in human rhinovirus 16-treated cells, demonstrating that Arpin is a key factor targeted to impair phagocytosis. We further show that Arpin is required for efficient uptake of multiple targets, for F-actin cup formation and for successful phagosome completion in macrophages. Interestingly, Arpin is recruited to sites of membrane extension and phagosome closure. Thus, we identify Arpin as a central actin regulator during phagocytosis that it is targeted by human rhinovirus 16, allowing the virus to perturb bacterial internalisation and phagocytosis in macrophages.

The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Dynamin-Actin Cross Talk Contributes to Phagosome Formation and Closure.

Phagocytosis is a mechanism used by macrophages to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force allowing plasma membrane extension around the particle. The closure step of phagocytosis, however, remains poorly defined. We used a dedicated experimental setup with Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor phagosome formation and closure in three dimensions in living cells. We show that dynamin-2, which mediates the scission of endocytic vesicles, was recruited early and concomitantly with actin during phagosome formation. Dynamin-2 accumulated at the site of phagosome closure in living macrophages. Inhibition of its activity with dominant negative mutants or drugs demonstrated that dynamin-2 is implicated in actin dynamics and pseudopod extension. Depolymerization of actin led to impaired dynamin-2 recruitment or activity. Finally, we show that dynamin-2 plays a critical role in the effective scission of the phagosome from the plasma membrane. Thus, we establish that a cross talk between actin and dynamin takes place for phagosome formation and closure before dynamin functions for scission.

Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments.

Phagocytosis in macrophages is receptor mediated and relies on actin polymerization coordinated with the focal delivery of intracellular membranes that is necessary for optimal phagocytosis of large particles. Here we show that phagocytosis by various receptors was inhibited in primary human macrophages infected with wild-type HIV-1 but not with a nef-deleted virus. We observed no major perturbation of F-actin accumulation, but adaptor protein 1 (AP1)-positive endosome recruitment was inhibited in HIV-1-infected cells. Expression of negative factor (Nef) was sufficient to inhibit phagocytosis, and myristoylation as well as the LL and DD motifs involved in association of Nef with AP complexes were important for this inhibition. We observed that Nef interferes with AP1 in association with membranes and/or with a cleaved regulatory form of AP1. Finally, an alteration of the recruitment of vesicle-associated membrane protein (VAMP3)- and tumor necrosis factor-alpha (TNFalpha)-positive recycling endosomes regulated by AP1, but not of VAMP7-positive late endosomes, was observed in phagocytic cups of HIV-1-infected macrophages. We conclude that HIV-1 impairs optimal phagosome formation through Nef-dependent perturbation of the endosomal remodeling relying on AP1. We therefore identified a mechanism of macrophage function down-regulation in infected cells.

Targeting CCR5 trafficking to inhibit HIV-1 infection.

Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.

HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger.

Human rhinovirus is frequently seen as an upper respiratory tract infection but growing evidence proves the virus can cause lower respiratory tract infections in patients with chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). In addition to airway epithelial cells, macrophages are crucial for regulating inflammatory responses to viral infections. However, the response of macrophages to HRV has not been analyzed in detail. We used in vitro monocyte-derived human macrophages to study the cytokine secretion of macrophages in response to the virus. Our results showed that macrophages were competent at responding to HRV, as a robust cytokine response was detected. However, after subsequent exposure to non-typeable Haemophilus influenzae (NTHi) or to LPS, HRV-treated macrophages secreted reduced levels of pro-inflammatory or regulatory cytokines. This "paralyzed" phenotype was not mimicked if the macrophages were pre-treated with LPS or CpG instead of the virus. These results begin to deepen our understanding into why patients with COPD show HRV-induced exacerbations and why they mount a defective response toward NTHi.

Adenomatous Polyposis Coli Defines Treg Differentiation and Anti-inflammatory Function through Microtubule-Mediated NFAT Localization.

Adenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT's nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth.

The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking.

Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1-infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1-infected macrophages, leading to strong alterations in phagolysosome biogenesis.

The NF-κB signaling protein Bcl10 regulates actin dynamics by controlling AP1 and OCRL-bearing vesicles.

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis.

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by alphaMbeta2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.