Macromolecular Structure underlying Recognition in Innate Immunity

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Temporal mRNA expression profile of cytokines produced by THP-1 cells incubated with different concentrations of properdin and Mycobacterium bovis BCG. (A) Pro-inflammatory cytokines: TNF-α, IL-1β, IL-6, and IL-12; (B) anti-inflammatory cytokines: TGF-β and IL-10. The expression of each cytokine was measured using qPCR, and the relative expression [relative quantification (RQ)] calculated by normalizing the data using human 18S rRNA expression as a control. The RQ value was calculated using the formula: RQ = 2−ΔΔCt. Assays were conducted twice in triplicates. Error bars represent SD. A two-way ANOVA test was performed on the data to determine significant differences in expression of cytokine production by properdin. All comparisons were significant (p < 0.05), unless where shown (ns, not significant, p > 0.05).
Original Research
08 May 2018
Human Properdin Modulates Macrophage: Mycobacterium bovis BCG Interaction via Thrombospondin Repeats 4 and 5
Maha Ahmed Al-Mozaini
10 more and 
Lubna Kouser

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen–macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1β, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-β) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1β, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host–pathogen interactions in tuberculosis.

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17 citations
(A) Properdin-coated carboxymethyl cellulose-coated carbon nanotube (CMC-CNT) show enhanced uptake by THP-1 cells. To observe internalization of carbon nanotubes (CNTs), PMA-differentiated THP-1 cells were treated with properdin or MBP-TSR4+5-coated biotinylated CMC-CNTs and uncoated biotinylated CMC-CNTs for 2 h. Cells were washed, fixed, permeabilized, and stained with Alexafluor-488-labeled Streptavidin (green) to reveal internalized biotin-CMC-CNTs (arrows). Alexafluor546-conjugated wheat germ agglutinin was used to reveal plasma membrane (red), and the nucleus was stained with Hoechst 33342 (Blue). Images shown are single sections (including side images) taken with a Nikon confocal microscope; scale bar, 20 µm arrow heads point to the CNTs adhering to the plasma membrane. (B) TSR4+5-coated CNTs show reduced uptake by THP-1 cells. To quantify the amount of uptake of protein coated or uncoated CMC-CNTs, THP-1 cells were incubated with 4 µg/mL of protein-coated biotin CMC-CNTs or uncoated CMC-CNTs for 2 h. The cells were lysed, and the amount of CNTs was quantified by an ELISA type assay. All experiments were done in triplicate; error bars represent mean ± SEM. A two-tailed, unpaired t-test was performed on the data to determine significant differences in uptake of CNTs between properdin-coated CNTs and TSR4+5-coated CNTs with CNTs only. All these comparisons were significant (P < 0.05), except where shown [not significant (ns), P > 0.05].
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29 citations
Original Research
24 March 2017
Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7
Isabell Hellmuth
14 more and 
Mark Helm
Application on commercial mono-alkyne-functionalized sense eGFP-siRNA. (A) Sense siRNA constructs for PBMC stimulation. (B) Denaturing polyacrylamide gel electrophoresis for comparison of the click efficiency of the single-stranded, alkyne-modified oligonucleotide MH662 (sense), free azides (4, 2, 5–8), and purified clicked (*) oligonucleotides, showing band shift after click reaction and additional fluorescent signals for SCy5 (blue) and PDI (purple). Before staining, excitation for dye-carrying constructs was done at 532 nm (PDI) and 633 nm (SCy5). Stains-all (gray/633 nm) was used as loading control to visualize non-fluorescent bands. Emission signals were recorded at 610BP30 nm and 670 nm (superimposition shown). (C) Titration of PBMCs with sense siRNA, clicked with molecules 2, 4, and 5–8 (nt, non-treated). IFN-α production was measured by ELISA as technical duplicate of biological triplicates (three donors). To account for donor variation in the absolute amount of IFN-α secreted, data from each individual were normalized to 1.0 μg/mL unmodified siRNA MH662 (=100%) of the respective donor (n = 3; mean + SD). (Asterisks above bars indicate P values evaluated by ANOVA and Sidak’s multiple comparisons test; no declaration = ns.) (D) eGFP-knockdown experiments with unlabeled control- (sense), TMA (6)- and PDI (8)-siRNA double strands [with antisense-strand MH533 (as)] in HeLa MAZ (stably expressing eGFP). IC50 (n = 3; mean + SD). (Respective P values for IC50 were evaluated by ANOVA and Dunnett’s multiple comparisons test.)

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

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9 citations
Melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) differentially induce the promoter activities of various IFNs. Ctenopharyngodon idella kidney cells seeded in 24-well plates were co-transfected with 380 ng pMDA5-HA or pRIG-I-HA, 380 ng pIFNpro-Luc (either pIFN1pro-Luc, pIFN2pro-Luc, pIFN3pro-Luc, pIFN4pro-Luc, pIFNγ1pro-Luc, or pIFNγ2pro-Luc), and 38 ng pRL-TK. Twenty-four hours later, one half of the cells were infected with grass carp reovirus, and the other half of the cells were treated with phosphate-buffered saline. Luciferase activities were detected at 6 h p.i. The remaining cell lysates were used for subsequent Western blotting tests to detect the overexpression of C. idella RIG-I-HA or CiMDA5-HA. Error bars represent ±SEMs obtained by testing each sample in quadruplicate. *P < 0.05; **P < 0.01.
Original Research
24 February 2017

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are critical cytosolic sensors that trigger the production of interferons (IFNs). Though their recognition functions are well identified, their unique roles in the downstream signal transduction remain to be elucidated. Herein, we report the differential effect between grass carp (Ctenopharyngodon idella) MDA5 (CiMDA5) and CiRIG-I on the production of various IFNs upon grass carp reovirus (GCRV) infection in C. idella kidney (CIK) cell line. In CIK cells, grass carp IFN1 (CiIFN1) and CiIFN3 are relatively highly expressed while CiIFN2 and CiIFN4 are relatively slightly expressed. Following GCRV infection, CiMDA5 induces a more extensive type I IFN response than CiRIG-I. Further investigation reveals that both CiMDA5 and CiRIG-I facilitate the expression and total phosphorylation levels of grass carp IFN regulatory factor (IRF) 3 (CiIRF3) and CiIRF7 upon GCRV infection or poly(I:C) stimulation. However, the difference is that CiRIG-I decreases the threonine phosphorylation level of CiIRF7. As a consequence, CiMDA5 enhances the heterodimerization of CiIRF3 and CiIRF7 and homodimerization of CiIRF7, whereas CiRIG-I facilitates the heterodimerization but attenuates homodimerization of CiIRF7. Moreover, the present study suggests that CiIRF3 and CiIRF7 heterodimers and CiIRF7 homodimers are able to induce more extensive IFN-I responses than CiIRF3 homodimers under GCRV infection. Additionally, CiMDA5 induces a stronger type II IFN (IFN-II) response against GCRV infection than CiRIG-I. Collectively, these results demonstrate that CiMDA5 plays a more potent role than CiRIG-I in IFN response to GCRV infection through differentially regulating the phosphorylation and dimerization of CiIRF3 and CiIRF7.

6,799 views
46 citations
Selected gene sets (sGS) were downregulated in rag1−/− egg embryos but were similar in early hatched larvae and both showed high mortalities when infected by spring viremia carp virus (SVCV). (A) Expression folds in embryo eggs 1 day after fertilization (n = 60). Reverse transcriptase and quantitative polymerase chain reaction data were first normalized by the formula, expression of each gene/expression of ef1a. Differential folds were then calculated by the formula, normalized expression of each rag1−/− gene/normalized expression of each rag1+/+ gene. Tbk1 fold was lower than 2−6 (not shown). Tnfa and nklysin were not done. Red dashed line, onefold boundary. *Statistically different from onefold with p < 0.05 by the t-test. (B) Expression folds in hatched larvae 3 days after fertilization (n = 45) calculated as in (A). (C) Kaplan–Meier survival curves of naïve rag1−/− (n = 90) and rag+/+ (n = 90) recently hatched larvae after bath infection in 104 pfu of SVCV per ml (n = 2) at 22°C (optimal replication for SVCV). Differences between rag1−/− and rag+/+ survival curves of SVCV-infected larvae as determined by the Gehan–Breslow–Wilcoxon test were significant with p < 0.05 (*). Solid lines, rag1−/− larvae. Dashed lines, rag+/+ larvae.
Original Research
13 February 2017

To investigate fish innate immunity, we have conducted organ and cell immune-related transcriptomic as well as immunohistologic analysis in mutant zebra fish (Danio rerio) lacking adaptive immunity (rag1−/−) at different developmental stages (egg, larvae, and adult), before and after infection with spring viremia carp virus (SVCV). The results revealed that, compared to immunocompetent zebra fish (rag1+/+), rag1−/− acquired increased resistance to SVCV with age, correlating with elevated transcript levels of immune genes in skin/fins and lymphoid organs (head kidney and spleen). Gene sets corresponding to apoptotic functions, immune-related multigene families, and interferon-related genes were constitutively upregulated in uninfected adult rag1−/− zebra fish. Overexpression of activated CASPASE-3 in different tissues before and after infection with SVCV further confirmed increased apoptotic function in rag1−/− zebra fish. Concurrently, staining of different tissue samples with a pan-leukocyte antibody marker showed abundant leukocyte infiltrations in SVCV-infected rag1−/− fish, coinciding with increased transcript expression of genes related to NK-cells and macrophages, suggesting that these genes played a key role in the enhanced immune response of rag1−/− zebra fish to SVCV lethal infection. Overall, we present evidence that indicates that rag1−/− zebra fish acquire an antiviral alert state while they reach adulthood in the absence of adaptive immunity. This antiviral state was characterized by (i) a more rapid response to viral infection, which resulted in increased survival, (ii) the involvement of NK-cell- and macrophage-mediated transcript responses rather than B- and/or T-cell dependent cells, and (iii) enhanced apoptosis, described here for the first time, as well as the similar modulation of multigene family/interferon-related genes previously associated to fish that survived lethal viral infections. From this and other studies, it might be concluded that some of the characteristics of mammalian trained immunity are present in lower vertebrates.

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41 citations
Original Research
13 December 2016
Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR
Lina Pednekar
11 more and 
Uday Kishore
ELISA to assess interaction between gC1qR and gh substitution mutants. Microtiter wells were coated with different quantities (0.25, 0.5, and 1 μg/well) of gC1qR. After blocking and washing, the wells were incubated with 2.5 μg/well of (A) ghA, R162A and R162E; (B) ghB, R114A, R114Q, R163A, R163E, R129A, R129E, T175L, and H117D; and (C) ghC, for 90 min at 37°C and 90 min at 4°C. Bound proteins were detected with anti-MBP monoclonal antibody followed by goat anti-mouse IgG–HRP conjugate. Data are representative of three experiments. (D) Ligand blot to show binding of ghA mutants R162A and R162E to gC1qR: PVDF membrane strips containing gC1qR were reacted with ghA, R162A and R162E, and then probed with anti-MBP monoclonal antibody followed by goat anti-mouse IgG–HRP.

The heterotrimeric globular head (gC1q) domain of human C1q is made up of the C-terminal ends of the three individual chains, ghA, ghB, and ghC. A candidate receptor for the gC1q domain is a multi-functional pattern recognition protein, gC1qR. Since understanding of gC1qR and gC1q interaction could provide an insight into the pleiotropic functions of gC1qR, this study was undertaken to identify the gC1qR-binding site on the gC1q domain, using the recombinant ghA, ghB, and ghC modules and their substitution mutants. Our results show that ghA, ghB, and ghC modules can interact with gC1qR independently, thus reinforcing the notion of modularity within the gC1q domain of human C1q. Mutational analysis revealed that while Arg162 in the ghA module is central to interaction between gC1qR and C1q, a single amino acid substitution (arginine to glutamate) in residue 114 of the ghB module resulted in enhanced binding. Expression of gC1qR and C1q in adherent monocytes with or without pro-inflammatory stimuli was also analyzed by qPCR; it showed an autocrine/paracrine basis of C1q and gC1qR interaction. Microscopic studies revealed that C1q and gC1qR are colocalized on PBMCs. Cell proliferation assays indicated that ghA, ghB, and ghC modules were able to attenuate phytohemagglutinin-stimulated proliferation of PBMCs. Addition of gC1qR had an additive effect on the anti-proliferative effect of globular head modules. In summary, our results identify residues involved in C1q-gC1qR interaction and explain, to a certain level, their involvement on the immune cell surface, which is relevant for C1q-induced functions including inflammation, infection, and immunity.

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16 citations
Original Research
02 March 2016
Structural and Functional Characterization of a Single-Chain Form of the Recognition Domain of Complement Protein C1q
Christophe Moreau
5 more and 
Nicole Thielens
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Complement C1q is a soluble pattern recognition molecule comprising six heterotrimeric subunits assembled from three polypeptide chains (A–C). Each heterotrimer forms a collagen-like stem prolonged by a globular recognition domain. These recognition domains sense a wide variety of ligands, including pathogens and altered-self components. Ligand recognition is either direct or mediated by immunoglobulins or pentraxins. Multivalent binding of C1q to its targets triggers immune effector mechanisms mediated via its collagen-like stems. The induced immune response includes activation of the classical complement pathway and enhancement of the phagocytosis of the recognized target. We report here, the first production of a single-chain recombinant form of human C1q globular region (C1q-scGR). The three monomers have been linked in tandem to generate a single continuous polypeptide, based on a strategy previously used for adiponectin, a protein structurally related to C1q. The resulting C1q-scGR protein was produced at high yield in stably transfected 293-F mammalian cells. Recombinant C1q-scGR was correctly folded, as demonstrated by its X-ray crystal structure solved at a resolution of 1.35 Å. Its interaction properties were assessed by surface plasmon resonance analysis using the following physiological C1q ligands: the receptor for C1q globular heads, the long pentraxin PTX3, calreticulin, and heparin. The 3D structure and the binding properties of C1q-scGR were similar to those of the three-chain fragment generated by collagenase digestion of serum-derived C1q. Comparison of the interaction properties of the fragments with those of native C1q provided insights into the avidity component associated with the hexameric assembly of C1q. The interest of this functional recombinant form of the recognition domains of C1q in basic research and its potential biomedical applications are discussed.

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26 citations