Mahdad Noursadeghi

HIV-1 Capsid-Cyclophilin Interactions Determine Nuclear Import Pathway, Integration Targeting and Replication Efficiency

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.

Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo

We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI‐2 type III secretion system is required for replication within splenic macrophages, and that sifA− mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI‐2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.

HIV-1 evades innate immune recognition through specific cofactor recruitment

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

HIV-1 infection of mononuclear phagocytic cells : the case for bacterial innate immune deficiency in AIDS

HIV-1 infection of mononuclear phagocytic cells, comprising monocytes, macrophages, and dendritic cells, has been the subject of extensive research over the past 20 years. The roles of mononuclear phagocytic cells in transmission of HIV-1 infection and as reservoirs of actively replicating virus have received particular attention. Experimental data have also accumulated about the effects of HIV-1 on the physiological function of mononuclear phagocytic cells, particularly their role in innate immunity to bacteria. The effects of HIV-1 on bacterial innate immune responses by mononuclear phagocytic cells are discussed here together with reports of direct interactions between HIV-encoded products and bacterial innate immune signalling pathways. These reports demonstrate mechanisms for HIV-mediated disruption of innate immune responses by mononuclear phagocytic cells that could provide novel therapeutic targets in HIV-infected patients. The clinical urgency is highlighted by greatly increased risk of invasive bacterial disease in this population, even in the era of highly active antiretroviral therapy. HIV-mediated injury to bacterial innate immunity provides an experimental paradigm that could broaden our overall understanding of innate immunity and be used to study responses to pathogens other than bacteria.

Towards host-directed therapies for tuberculosis

The treatment of tuberculosis is based on combinations of drugs that directly target Mycobacterium tuberculosis. A new global initiative is now focusing on a complementary approach of developing adjunct host-directed therapies.

HIV-1 infection of macrophages is dependent on evasion of innate immune cellular activation.

Objective:The cellular innate immune response to HIV-1 is poorly characterized. In view of HIV-1 tropism for macrophages, which can be activated via pattern recognition receptors to trigger antimicrobial defences, we investigated innate immune responses to HIV-1 by monocyte-derived macrophages. Design:In a model of productive HIV-1 infection, cellular innate immune responses to HIV-1 were investigated, at the level of transcription factor activation, specific gene expression and genome-wide transcriptional profiling. In addition, the viral determinants of macrophage responses and the physiological effect of innate immune cellular activation on HIV-1 replication were assessed. Results:Productive HIV-1 infection did not activate nuclear factor-κB and interferon regulatory factor 3 transcription factors or interferon gene expression (IFN) and caused remarkably small changes to the host-cell transcriptome, with no evidence of inflammatory or IFN signatures. Evasion of IFN induction was not dependent on HIV-1 envelope-mediated cellular entry, inhibition by accessory proteins or reverse transcription of ssRNA that may reduce innate immune cellular activation by viral RNA. Furthermore, IFNβ priming did not sensitize responses to HIV-1. Importantly, exogenous IFNβ or stimulation with the RNA analogue poly I:C to simulate innate immune activation invoked HIV-1 restriction. Conclusion:We conclude that macrophages lack functional pattern recognition receptors for this virus and that HIV-1 tropism for macrophages helps to establish a foothold in the host without triggering innate immune cellular activation, which would otherwise block viral infection effectively.

Production of granulocyte colony-stimulating factor in the nonspecific acute phase response enhances host resistance to bacterial infection

Mice mounting an acute phase response, induced by sterile inflammation after a single s.c. injection of casein 24 h beforehand, were remarkably protected against lethal infection with Gram-positive or Gram-negative bacteria. This was associated with enhanced early clearance of bacteremia, greater phagocytosis and oxidative burst responses by neutrophils, and enhanced recruitment of neutrophils into tissues compared with control, nonacute phase mice. Casein-induced inflammation was also associated with increased concentrations of G-CSF in serum, and administration of neutralizing Ab to this cytokine completely abrogated protection against Escherichia coli infection after casein pretreatment. Injection of recombinant murine G-CSF between 3 and 24 h before infection conferred the same protection as casein injection. In contrast, the casein-induced acute phase response affected neither serum values of TNF-α, IL-1β, or IL-6 after E. coli infection nor susceptibility to LPS toxicity. Furthermore, protection against infection was unaffected in IL-1R knockout mice, which have deficient acute phase plasma protein responses, or after nonspecific inhibition of acute phase protein synthesis by d-galactosamine or specific depletion of complement C3 by cobra venom factor. Increased production of G-CSF in the acute phase response is thus a key physiological component of host defense, and pretreatment with G-CSF to prevent bacterial infection in at-risk patients now merits further study, especially in view of increasing bacterial resistance to antibiotics.

Streptococcus pneumoniae Capsular Serotype Invasiveness Correlates with the Degree of Factor H Binding and Opsonization with C3b/iC3b

ABSTRACT Different capsular serotypes of Streptococcus pneumoniae vary markedly in their ability to cause invasive infection, but the reasons why are not known. As immunity to S. pneumoniae infection is highly complement dependent, variations in sensitivity to complement between S. pneumoniae capsular serotypes could affect invasiveness. We have used 20 capsule-switched variants of strain TIGR4 to investigate whether differences in the binding of the alternative pathway inhibitor factor H (FH) could be one mechanism causing variations in complement resistance and invasive potential between capsular serotypes. Flow cytometry assays were used to assess complement factor binding and complement-dependent neutrophil association for the TIGR4 capsule-switched strains. FH binding varied with the serotype and inversely correlated with the results of factor B binding, C3b/iC3b deposition, and neutrophil association. Differences between strains in FH binding were lost when assays were repeated with pspC mutant strains, and loss of PspC also reduced differences in C3b/iC3b deposition between strains. Median FH binding was high in capsule-switched mutant strains expressing more invasive serotypes, and a principal component analysis demonstrated a strong correlation between serotype invasiveness, high FH binding, and resistance to complement and neutrophil association. Further data obtained with 33 clinical strains also demonstrated that FH binding negatively correlated with C3b/iC3b deposition and that median FH binding was high in strains expressing more invasive serotypes. These data suggest that variations in complement resistance between S. pneumoniae strains and the association of a serotype with invasiveness could be related to capsular serotype effects on FH binding.

Increased Susceptibility of C1q-Deficient Mice to Salmonella enterica Serovar Typhimurium Infection

ABSTRACT The role of the complement system in host defense against Salmonella infection is poorly defined. Bacterial cell wall O-antigen polysaccharide can activate the alternative pathway in vitro. No studies, however, have elucidated the role of the classical pathway in immunity to Salmonella spp. in vivo. C1q-deficient mice (C1qa−/−) on a 129/Sv genetic background and strain-matched controls were infected intraperitoneally and intravenously with Salmonella enterica serovar Typhimurium and monitored over a 14-day period. After inoculation by either route, the C1qa−/− mice were found to be significantly more susceptible to Salmonella infection. Hepatic and splenic bacterial counts, performed at various time points, showed increased numbers of colonies in complement-deficient mice compared to controls. Analysis of blood clearance showed no difference between the two experimental groups during the first 15 min. However, after 20 min and until 6 h postinfection, numbers of circulating bacteria were significantly higher in complement-deficient mice. In vitro experiments using either resident or thioglycolate-elicited peritoneal macrophages showed a significant increase in the number of bacteria inside C1q-deficient macrophages compared to controls irrespective of the serum used for opsonizing the bacteria. These findings could not be explained either by an increased bacterial uptake, analyzed in vitro and in vivo using green fluorescent protein-tagged salmonellae, or by a defect in the respiratory burst or in NO production. The data presented here suggest the possibility of novel pathways by which C1q may modulate the pathogenesis of infectious diseases caused by intracellular pathogens.

Cerebrospinal fluid cytokine profiles predict risk of early mortality and immune reconstitution inflammatory syndrome in HIV-associated cryptococcal meningitis.

Understanding the host immune response during cryptococcal meningitis (CM) is of critical importance for the development of immunomodulatory therapies. We profiled the cerebrospinal fluid (CSF) immune-response in ninety patients with HIV-associated CM, and examined associations between immune phenotype and clinical outcome. CSF cytokine, chemokine, and macrophage activation marker concentrations were assayed at disease presentation, and associations between these parameters and microbiological and clinical outcomes were examined using principal component analysis (PCA). PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting primarily of Th1, Th2, and Th17-type cytokines. The presence of this CSF cytokine response was associated with evidence of increased macrophage activation, more rapid clearance of Cryptococci from CSF, and survival at 2 weeks. The key components of this protective immune-response were interleukin (IL)-6 and interferon-γ, IL-4, IL-10 and IL-17 levels also made a modest positive contribution to the PC1 score. A second component of co-correlated chemokines was identified by PCA, consisting primarily of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). High CSF chemokine concentrations were associated with low peripheral CD4 cell counts and CSF lymphocyte counts and were predictive of immune reconstitution inflammatory syndrome (IRIS). In conclusion CSF cytokine and chemokine profiles predict risk of early mortality and IRIS in HIV-associated CM. We speculate that the presence of even minimal Cryptococcus-specific Th1-type CD4+ T-cell responses lead to increased recruitment of circulating lymphocytes and monocytes into the central nervous system (CNS), more effective activation of CNS macrophages and microglial cells, and faster organism clearance; while high CNS chemokine levels may predispose to over recruitment or inappropriate recruitment of immune cells to the CNS and IRIS following peripheral immune reconstitution with ART. These results provide a rational basis for future studies of immune modulation in CM, and demonstrate the potential of baseline immune profiling to identify CM patients most at risk of mortality and subsequent IRIS.