Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections.

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide’s and Fluspirilene’s efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Setting a course for intervening in host-pathogen interactions.

The 25th Dageraad Symposium on The Molecular Mechanisms of Host-Pathogen Interactions in Infectious Disease: Towards Better Intervention Strategies? was held in Enkhuizen, The Netherlands, from 29 June to 3 July 2001.

Conformational alterations during biosynthesis of HLA-DR3 molecules controlled by invariant chain and HLA-DM.

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.

Human deficiencies in type 1 cytokine receptors reveal the essential role of type 1 cytokines in immunity to intracellular bacteria.

Studies on patients with idiopathic, severe infections due to poorly pathogenic mycobacteria and Salmonella have revealed that many of these patients are unable to produce or respond to interferon-gamma (IFN-gamma). This inability results from causative, deleterious genetic mutations in either one of four different genes in the type 1 cytokine cascade, encoding interleukin-12Rbeta1 (IL-12Rbeta1), IL-12p40, IFN-gammaR1 or IFN-gammaR2. The immunological phenotypes resulting from the seven groups of complete or partial deficiencies in type 1 cytokine (receptor) genes that have been distinguished thus far will be summarized and discussed, and placed in a broader context in relation to disease susceptibility.

Residual type 1 immunity in patients genetically deficient for interleukin 12 receptor beta1 (IL-12Rbeta1): evidence for an IL-12Rbeta1-independent pathway of IL-12 responsiveness in human T cells.

Genetic lack of interleukin 12 receptor beta1 (IL-12Rbeta1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-gamma production. To study IL-12Rbeta1-independent residual IFN-gamma production, we have generated mycobacterium-specific T cell clones (TCCs) from IL-12Rbeta1-deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-gamma production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rbeta2 was found to be normally expressed in the absence of IL-12Rbeta1, and could be upregulated by IFN-alpha. Expression of IL-12Rbeta2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-alpha/IFN-alphaR ligation resulted in Stat4 activation in both control and IL-12Rbeta1-deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin alpha6, and IL-12Rbeta2 on IL-12Rbeta1-deficient cells, whereas this was normal on control cells. IL-12-induced IFN-gamma production in IL-12Rbeta1-deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway.Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rbeta1. Moreover, the results reveal the presence of a novel IL-12Rbeta1/Stat4-independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rbeta1 deficiency.

Cryptic open reading frames in plasmid vector backbone sequences can provide highly immunogenic cytotoxic T-lymphocyte epitopes.

Murine tumor cells obtained through transfection of expression plasmids carrying activated cellular and/or viral oncogenes constitute formidable tools for immunological tumor research. As reported previously, mouse embryo cells of C57BL/6 origin, transformed by mutated p53 or human papilloma virus type 16 (HPV16), present, at their surface, MHC-bound peptides that are derived from the p53 and the HPV16 E7 oncoproteins, respectively, which can serve as a target for a highly effective antitumor T-cell response. Here, we describe the identification, through molecular cloning, of an additional, highly immunodominant peptide that is presented by the aforementioned HPV16- and p53-transformed cells. This peptide is encoded by a cryptic open reading frame in the backbone sequences of the plasmids that had been used to generate these cells. Considerable amounts of transcripts encompassing this open reading frame were detected in the cells concerned. These transcripts were the result of the bidirectional nature of the retroviral long terminal repeat (LTR) present in the expression plasmids used for transfection, which resulted in transcription of the gene of interest, as well as in transcription of the vector sequences positioned at the other side of the LTR. Due to this mechanism, all tumor cells harboring LTR-driven expression plasmids expressed the highly immunogenic peptide, whereas cells containing plasmids driven by more unidirectional promoters exhibited lower levels of this peptide. LTR-driven expression plasmids were also shown to encode this peptide epitope when used for DNA vaccination, as mice vaccinated with such a plasmid developed a CTL response against this peptide. Our data show that awareness of plasmid backbone-derived epitopes is of crucial importance for the correct interpretation of preclinical experiments and for the design of DNA vaccines.

The generation of SDS-stable HLA DR dimers is independent of efficient peptide binding.

MHC class II molecules can exist in two conformations which can be distinguished on the basis of their stability in SDS. The formation of SDS-stable dimers has been shown to correlate with persistent expression of antigen MHC class II-peptide complexes by murine antigen-presenting cells. HLA DR molecules contain either a Val or a Gly at position 86 of the beta chain, which is located in a conserved and prominent hydrophobic pocket in the peptide binding site. Here we show that Val86-containing DR molecules more frequently select peptides which induce the formation of SDS-stable dimers than Gly86 variants. Using analogues of the influence virus haemagglutinin epitope 307-319 we found that the replacement of the aromatic hydrophobic anchor residue (Tyr)at position 309 by amino acids with an aliphatic hydrophobic side chain resulted in the specific formation of high numbers of SDS-stable Val86-DR but not Gly86-DR dimers. These results indicate that the fit between the first anchor residue and the hydrophobic pocket around Dr beta 86 plays a critical role in the formation of SDS-stable DR dimers. Synthetic analogues of naturally processed DR-associated peptides displayed promiscuity in their capacity to bind to several DR specificities and in their ability to induce the SDS-stable conformation. However, no correlation was observed between binding capacity and the ability to induce the SDS-stable conformation. Since it has been shown that SDS stability can relate to the kinetics of peptide-MHC class II interactions, the definition of the requirements for the formation of SDS-stable HLA class II molecules may be important for the design of effective peptide-based immunomodulation protocols.

DR4Dw4/DR53 molecules contain a peptide from the autoantigen calreticulin.

Rheumatoid arthritis (RA) occurs more frequently in HLA-DR4+ individuals than in those who do not express this MHC class II molecule. Although the role of this genetic factor in the immunopathology of this autoimmune disease is unclear, the association of RA with HLA-DR4 may indicate that DR4 molecules present autoantigen(s) to T cells. Here we report the analysis of naturally processed peptides, eluted from a mixture of HLA-DR4Dw4 (DRB1*0401) and DR53 (DRB4*0101) molecules isolated from an RA patient-derived EBV-transformed B cell line. Several (size variants of) self-peptides originating from the autologous molecules HLA-A2, HLA-Cw9, HLA-B62, HLA-DR4Dw4 and HLA-DR53, were identified. We also found a sequence that has no homology to any protein in the SwissProt protein sequence databank, and a peptide identical to an internal fragment of the autoantigen calreticulin. The association of the identified peptides with cells expressing HLA-DR4Dw4/DR53 was confirmed by peptide binding analysis. In agreement with previously described peptide binding motifs for DR4Dw4, most peptides contained an aromatic residue (Phe, Tyr, Trp) at relative position i and a small hydroxyl-containing residue (Ser, Thr) at i + 5. Our findings indicate that in RA patient-derived EBV-transformed B cells DR4Dw4/DR53 molecules present a peptide from the autoantigen calreticulin. Interestingly, autoantibodies against calreticulin have been found in various rheumatic diseases, including rheumatoid arthritis. Thus, the analysis of HLA class II-bound peptides can lead to the identification of putative T helper epitopes, which might be involved in the immunopathology of autoimmune diseases.

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing.

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogenous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.

HLA-DR beta chain residue 86 controls DR alpha beta dimer stability.

Major histocompatibility complex class II molecules exist in two forms, which can be distinguished on the basis of their stability in sodium dodecyl sulfate (SDS) as SDS-stable and SDS-unstable alpha beta dimers. The ratio of stable vs. unstable alpha beta dimers varies between murine H-2 alleles and isotypes, but the molecular basis for this observation is unknown. Here we show that for the human HLA-DRB1 and HLA-DRB3 gene products this ratio is controlled by the valine/glycine dimorphism at position 86. Haplotypes coding for DR beta chains with a valine at position 86 express higher numbers of stable dimers compared to similar haplotypes expressing DR beta chains with a glycine at that position. Reverse-phase high-performance liquid chromatography analysis of iodinated peptides, which were eluted from DR dimers with either a DRB1*1101 or a DRB1*1104 beta chain which differ only at position 86, indicated that these DR dimers contain (partially) distinct sets of peptides. The valine/glycine dimorphism is highly conserved, present in most HLA-DR alleles and influences peptide-binding. Analysis of the occurrence of the Val86 and the Gly86 gene products revealed that these are not equally present in the population. Depending on the DR specificity either the Val86 of Gly86 allelic variant is favored. Thus, the natural, highly conserved dimorphism at HLA-DR beta chain position 86 influences peptide selection. The dimorphism is therefore likely to influence antigen presentation and forms the molecular basis for the observed differences in stability of Val86- and Gly86-containing DR dimers in the presence of SDS.