Abstract Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts cocultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to up-regulation of collagen. Direct contact between macrophages and fibroblasts was not required for collagen up-regulation. Macrophages produced TGF-β following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant up-regulation of collagen. Simultaneously, the levels of TGF-β-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI down-regulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and up-regulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or small interfering RNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas small interfering RNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to the down-regulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen.
ABSTRACT IL-4δ2 is a natural splice variant of IL-4 that lacks the region encoded by the second exon. Numerous reports have suggested that the expression levels of IL-4δ2 change in various diseases, especially those with pulmonary involvement, but the in vivo effects of this splice variant have never been studied. Replication-deficient, AdV-mediated gene delivery of mIL-4δ2 to mouse lungs in vivo was used, and the effects compared with similar adenoviral delivery of mIL-4 or with infection with a noncoding NULL viral construct. Overexpression of IL-4δ2 or IL-4 caused pulmonary infiltration by T and B lymphocytes, whereas in contrast to IL-4, IL-4δ2 did not induce eosinophilia or goblet cell hyperplasia. Microarray analysis of global gene expression revealed that IL-4δ2 and IL-4 had differential effects on gene expression. These splice variants also differentially regulated pulmonary levels of the cytokines TNF-α, eotaxin, IL-1α, IFN-γ, and MCP-1, whereas both tended to increase total lung collagen modestly. Pulmonary infiltration by lymphocytes in response to overexpression of IL-4δ2 was attenuated but not abrogated completely by germline deficiency of IL-4Rα or STAT6, whereas deficiency of endogenous IL-4 had no effect. Thus, IL-4δ2 promotes lymphocytic inflammation in vivo (although differentially from IL-4, in part), and the effects of IL-4δ2 are not mediated by endogenous IL-4. Differential targeting of IL-4δ2 and IL-4 may therefore be considered in developing future therapeutic agents.
Abstract Placental malaria vaccines (PMV) are being developed to prevent severe sequelae of placental malaria (PM) in pregnant women and their offspring. The leading candidate vaccine antigen VAR2CSA mediates parasite binding to placental receptor chondroitin sulfate A (CSA). Despite promising results in small animal studies, recent human trials of the first two PMV candidates (PAMVAC and PRIMVAC) generated limited cross-reactivity and cross-inhibitory activity to heterologous parasites. Here, we immunized Aotus nancymaae monkeys with three PMV candidates (PAMVAC, PRIMVAC and ID1-ID2a_M1010) adjuvanted with Alhydrogel®, and exploited the model to investigate boosting of functional vaccine responses during PM episodes as well as with nanoparticle antigens. PMV candidates induced high levels of antigen-specific IgG with significant cross-reactivity across PMV antigens by ELISA. Conversely, PMV antibodies recognized native VAR2CSA and blocked CSA-adhesion of only homologous parasites and not heterologous parasites. PM episodes did not significantly boost VAR2CSA antibody levels or serum functional activity; nanoparticle and monomer antigens alike boosted serum reactivity but not functional activities. Overall, PMV candidates induced functional antibodies with limited heterologous activity in Aotus monkeys, similar to responses reported in humans. The Aotus model appears suitable for preclinical down-selection of PMV candidates and assessment of antibody boosting by PM episodes. Research in Context Evidence before this study The Plasmodium falciparum erythrocyte membrane protein VAR2CSA is the leading vaccine candidate antigen to protect pregnant women against placental malaria (PM), which causes serious adverse pregnancy outcomes particularly in first-time mothers living in malaria-endemic areas. Two VAR2CSA-based vaccines (PAMVAC and PRIMVAC) induced strong heterologous functional antibodies in small animals, but induced antibodies with limited cross-inhibitory functional activity in human clinical trials. These observations highlighted the need to establish new animal models that could better recapitulate human pathogenesis and immunity. In ongoing development of a nonhuman primate model for PM, we established an Aotus nancymaae model susceptible to P. falciparum infection during pregnancy that reproduces all the immunoparasitological and histological features of human PM. In this study, we explore the new Aotus model as a platform for evaluating PM vaccine (PMV) immunogenicity and for boosting of vaccine responses during PM episodes. Added value of this study In this manuscript, we demonstrate that PMV (including PAMVAC and PRIMVAC) are immunogenic in Aotus monkeys, inducing antibodies with mainly homologous and little heterologous functional activity, as seen in humans but contrary to preclinical reports on these vaccines in small animals. Implications of all the available evidence Our findings suggest Aotus is a suitable model to assess immunogenicity of VAR2CSA-derived vaccines, in contrast to small animal models. PMV data from human trials and Aotus monkeys suggest that improvements to current VAR2CSA immunogens and/or adjuvants are needed to enhance protective antibody responses, as are studies that evaluate the potential for natural infection to boost vaccine antibody in pregnancy. Therefore, the Aotus PM model may be useful to assess second-generation PMVs seeking to increase strain-transcending activity and to prioritize these for further clinical development.
Abstract Information about the cognate pairing of TCR alpha-beta chains and BCR IgH and IgKL chains encoded by individual T and B cells is key to understanding adaptive immune responses and developing therapeutic applications. We have previously reported the development of a sensitive technology that allows the amplification and identification of the paired human TCR alpha and beta chains from single T cells, termed iPair-TCR. Here, we report the extension of this technology to identify paired human BCR IgH and IgKL chains from antigen-specific single B cells. In this proof of concept study, we identified paired-VDJ-receptors from antigen-specific B-cells from nine Pfs230-EPA immunized Malian adults using the iPair-BCR method. Next, we developed a method to rapidly generate Fab fragments and demonstrate the binding of several of these single cell targets to the original Pfs230 antigen. Single cells of interest were identified based upon their repeated frequency on the plate, which indicates a clonal selection. The corresponding heavy and light chains were PCR amplified from selected wells. Using overlap extension PCR, all necessary elements for in vitro transcription and translation and either the CH1 or C-kappa-domain were added to both the 5′ and 3′ ends of the single cell VDJ. After in vitro transcription and translation, four out of five tested Fab fragments demonstrated binding through a colorimetric ELISA assay. The overall process after VDJ identification can be performed in under a week indicating the utility of our technology for rapid identification of antigen-specific BCRs and functional binding characteristics.
We previously described a natural splice variant of IL‐4 that lacks exon 2, and reported that recombinant IL‐4δ2 antagonizes functional effects of IL‐4 on cultured T and B lymphocytes and monocytes. Changes in the absolute and relative expression levels of IL‐4 and IL‐4δ2 have been reported in the lungs of patients with asthma, tuberculosis, and HIV infection. Functional effects of IL‐4δ2 in vivo have not been characterized. Adenoviral gene delivery of IL‐4 or IL‐4δ2 to mouse lungs in vivo in comparison with AdV‐NULL infections was utilized. Overexpression of IL‐4 or IL‐4δ2 was confirmed by ELISA and LC/MS. Delivery of either IL‐4 or IL‐4δ2 caused profound peribronchial and perivascular infiltration of mostly T cell, equally CD4+ and CD8+, with some granulocytes, macrophages, and plasma cells. In the bronchoalveolar lavage cells, the relative contribution of lymphocytes changed from normal 1.7±1.2% to 33±8% in IL‐4‐expressing and 53±6% in IL‐4δ2‐expressing mice. Less than 1% of the infiltrating CD8+ cells but 17±3% of CD4+ cells and were also CD25+. These changes peaked at 7–14 days following the infections, and declined by 21–28 days. The implication based on experiments in cell culture that IL‐4δ2 is simply a passive inhibitor of IL‐4 effects is likely incorrect. The splice variant IL‐4δ2 is an active cytokine mediating pulmonary infiltration of mostly T cells in vivo. Support: R01HL074067, VA Merit (SPA).
Placental malaria vaccines (PMVs) are being developed to prevent severe sequelae of placental malaria (PM) in pregnant women and their offspring. The leading candidate vaccine antigen VAR2CSA mediates parasite binding to placental receptor chondroitin sulfate A (CSA). Despite promising results in small animal studies, recent human trials of the first two PMV candidates (PAMVAC and PRIMVAC) generated limited cross-reactivity and cross-inhibitory activity to heterologous parasites. Here we immunized Aotus nancymaae monkeys with three PMV candidates (PAMVAC, PRIMVAC and ID1-ID2a_M1010) adjuvanted with Alhydrogel, and exploited the model to investigate boosting of functional vaccine responses during PM episodes as well as with nanoparticle antigens. PMV candidates induced high levels of antigen-specific IgG with significant cross-reactivity across PMV antigens by enzyme-linked immunosorbent assay. Conversely, PMV antibodies recognized native VAR2CSA and blocked CSA adhesion of only homologous parasites and not of heterologous parasites. PM episodes did not significantly boost VAR2CSA antibody levels or serum functional activity; nanoparticle and monomer antigens alike boosted serum reactivity but not functional activities. Overall, PMV candidates induced functional antibodies with limited heterologous activity in Aotus monkeys, similar to responses reported in humans. The Aotus model appears suitable for preclinical downselection of PMV candidates and assessment of antibody boosting by PM episodes.
T cells are commonly involved in lung fibrosis, but their mechanistic role is unclear. Lung T cells from patients with scleroderma, idiopathic pulmonary fibrosis, and healthy controls were studied. CCL18 gene delivery in vivo was used to induce selective chemoattraction of T cells to otherwise healthy or bleomycin‐injured mouse lungs. The fibrotic potential of T cells was studied in cell culture. Immunohistologically and flowcytometrically, integrins αVβ3 and αVβ5 were expressed on 32±15% of T cells in patients with fibrosis but not in healthy controls; higher integrin expression levels correlated with worse pulmonary function measures. Levels of mRNAs for these integrins were also increased. Infiltration of T cell in the otherwise healthy mouse lungs was accompanied by integrin expression on T cells, elevated active TGF‐β, and T cell‐dependent collagen accumulation. In contrast, CCL18‐driven infiltration of T cells combined with bleomycin injury showed lack of integrin expression on T cells, lower active TGF‐β, and partial inhibition of collagen accumulation. In vitro overexpression of integrin αVβ3 or αVβ5 on Jurkat cells caused an increase in TGF‐β and collagen in co‐cultures with primary lung fibroblasts. In conclusion, T cells may act pro‐ or antifibrotically, by expressing or not expressing integrins αVβ3 and/or αVβ5, and likely activating or not activating TGF‐β. Support: R01HL074067, VA Merit (SPA).
A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCalpha, PKCdelta, and PKCepsilon), two isoforms (PKCdelta and PKCepsilon) have been implicated in profibrotic intracellular signaling. The role of PKCalpha-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCalpha was found mostly in the cytoplasm, whereas PKCdelta and PKCepsilon were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCalpha but not PKCdelta or PKCepsilon underwent rapid (within 5-10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCalpha and ERK2, but not PKCdelta or PKCepsilon, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCalpha Gö6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCalpha signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCalpha nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCalpha but not PKCdelta or PKCepsilon mediate the profibrotic effect of CCL18. PKCalpha may therefore become a viable target for future antifibrotic therapies.
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