Infection of immune competent macrophages expressing functional Slc11a1 alters global gene expression, regulation of metal ions, and infection outcomes

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron availability, increasing exposure to bactericidal copper, and manipulating zinc to restrict the growth of pathogens. Manipulation of zinc at the host-pathogen interface depends on both the pathogen’s identity and the nature of the host cell. Here we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella Typhimurium. Unlike Balb/c and C57BL/6 mice, 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 Hrs and 18 Hrs post-infection and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 36-fold 12 hrs post-infection. Further, manipulation of zinc in the media alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing our results to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response, with 129S6 macrophages transitioning from M1 to M2 polarization over the course of infection and showing signs of recovery. Our results reveal that functional Slc11a1 profoundly affects the transcriptional landscape upon infection. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. 129S6 macrophage leverage intricate means of manipulating zinc availability and distribution to limit the pathogen’s access to zinc while simultaneously ensuring sufficient zinc to support the immune response. Author summary Metal ions play an important role in influencing how immune cells such as macrophages respond to infection by pathogens. Because metal ions are both essential to survival, as well toxic when present is excessive amounts, the host and the pathogen have evolved diverse strategies to regulate metal acquisition and availability. Here, we show that the metal transporter slc11a1 plays a critical role in defining the host response to Salmonella infection. Infection causes widespread changes in expression of metal regulatory genes to limit the pathogen’s access to iron, increase its exposure to copper, and remodel zinc to ensure increased zinc in the cytosol and limited zinc for the pathogen. Macrophages expressing functional slc11a1 have a different profile of metal regulation and vastly different outcomes compared to immune compromised macrophage, demonstrating significantly different nutritional immune responses in immune competent versus immune compromised macrophages.

141 exposure and subjected to global RNA sequencing (Fig 1A). Principal component analysis shows 142 that samples clustered predominantly by time post infection and status of bacteria (alive versus 143 heat killed, Fig 1B). 144 We identified 7766 genes that were differentially expressed and performed hierarchical 145 clustering to examine the main patterns of gene expression changes. We limited analysis to 146 genes with significant differential expression (adjusted p-value (padj) < 0.01), as determined by 147 a DESeq2 log ratio test. An expression level cutoff was also applied (see Methods). An  Fig S1D). These results indicate that the M2 anti-inflammatory response along 172 with many metal associated genes are upregulated over time in response to infection, 173 regardless of whether the bacteria are alive or HK.

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The genes in group 2 (Fig 2B) are upregulated at 2 Hrs, with higher expression in alive 175 compared to HK conditions, and are subsequently suppressed below baseline at 18 Hrs. This 176 group is enriched in proinflammatory M1 immune genes(31), including tnf, the inflammasome 177 component nlrp3, and irf1 responsible for activating cytokine production. This group also 178 contains multiple genes involved in membrane transport of zinc (slc30a4, slc39a6/a8/a10).
179 Another hallmark proinflammatory M1 marker tlr2 and metal regulatory genes (the zinc-180 dependent transcription factor mtf1 and iron storage ferritin light chain ftl1) are present in 181 group 3 (Supplementary Fig S1A,

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There are also significant changes in transporters dedicated to import, export, and 279 distribution of Fe, Cu and Zn (Fig 5B). There is a downregulation of the transferrin (trf) Fe

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In addition to metal transport, there are significant changes in genes that are metal 294 dependent (Fig 5C) and genes related to metal homeostasis (Fig 5D). There is an increase in

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In addition to measuring bacterial clearance, the pDiGc plasmid can also be used to 388 measure bacterial replication, as the DsRed signal is diluted with every cell division. We 389 compared the DsRed signal versus GFP signal as a function of time and different media 390 conditions (Fig 8). In standard macrophage growth media (top row), the DsRed signal decreases

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To evaluate potential differences in genes involved in nutritional immunity between the 416 two datasets, we examined all genes with UNIPROT keywords for iron, zinc, or copper transport 417 or homeostasis. 418 There are a number of similarities in regulation of metal-transport, -homeostasis, and -419 dependent genes, indicating that there are common mechanisms for managing metal ions at 420 the host-pathogen interface, even when there are differences in the host. In particular, there is 421 a significant decrease in Fe import (trf), export (slc40a1), and an increase in ferritin storage 422 (fth1) in both studies (Fig 9A). Similarly, both studies show upregulation of plasma membrane 423 copper transporter slc31a1 indicating Cu uptake from the extracellular space (Fig 9B).

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There are also notable differences in infection-induced expression changes in genes 446 encoding Zn transporters and Zn-dependent proteins between the two systems (Fig 9C, D).  slc39a7, slc30a4, slc30a5). Increased expression of mt1 and mt2 genes, which encode 496 Zn buffering proteins, further suggests an increase in cytosolic Zn. We also observed numerous 497 changes in metal-dependent proteins and enzymes, many of which play an important role in 498 nutritional immunity (lcn2, ltf, cp, steap3, sod2, arg1, nos2, mmp, car, s100a8). Some of these 499 changes have been seen in a variety of mouse model systems in response to diverse 500 intracellular pathogens, suggesting widespread or universal strategies for the host to combat 501 infection. In particular, the changes in gene expression to limit Fe(6,51) and accumulate Cu 502 appear to be common strategies for nutritional immunity(4). Indeed, the changes in expression  570 identified an RNAseq study that was carried out in macrophages from C57BL/6 mice(47), with 571 strong parallels to our conditions, and reanalyzed the data along with ours in an identical 572 pipeline. We found that 4500 genes were differentially expressed in one study but not the 573 other, with differences in expression at baseline as well as in response to infection. This is 574 consistent with studies of mouse lines possessing one or two functional slc11a1 alleles on a 575 C57BL/6 background(30,49). These mice experienced lower infection loads but still ultimately 576 succumbed to Salmonella infection, implying differences between C57BL/6 mice and 129S6 577 mice that extend beyond Slc11a1. However, the magnitude of differential expression was 578 unexpected, as it involved more than a third of all genes differentially expressed in our data.
579 Annotation analysis of this differential gene list indicates that these cell types tend toward  Figure S7). This is in contrast to findings in Slc11a1 non-621 functional macrophages which indicate that dividing Salmonella may actively induce M2 622 polarization to enhance bacterial survival, and that macrophages exposed to but not infected  (Fig 9)