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. 2021 Nov;226(6):152150.
doi: 10.1016/j.imbio.2021.152150. Epub 2021 Oct 25.

Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages

Eric Yau  1 , Yan Chen  2 , Chunhua Song  3 , Jason Webb  4 , Marykate Carillo  4 , Yuka Imamura Kawasawa  5 , Zhenyuan Tang  6 , Yoshinori Takahashi  6 , Todd M Umstead  4 , Sinisa Dovat  6 , Zissis C Chroneos  7
Affiliations

Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages

Eric Yau et al. Immunobiology. 2021 Nov.

Abstract

Macrophages play an important role in maintaining tissue homeostasis, from regulating the inflammatory response to pathogens to resolving inflammation and aiding tissue repair. The surfactant protein A (SP-A) receptor SP-R210 (MYO18A) has been shown to affect basal and inflammatory macrophage states. Specifically, disruption of the longer splice isoform SP-R210L/MYO18Aα renders macrophages hyper-inflammatory, although the mechanism by which this occurs is not well understood. We asked whether disruption of the L isoform led to the hyper-inflammatory state via alteration of global genomic responses. RNA sequencing analysis of L isoform-deficient macrophages (SP-R210L(DN)) revealed basal and influenza-induced upregulation of genes associated with inflammatory pathways, such as TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockout of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) cells, with increased representation around genes relevant to inflammatory pathways. Additional ChIP-seq analysis of histone H3 methylation marks showed decreases in both repressive H3K9me3 and H3K27me3 marks with a commensurate increase in transcriptionally active (H3K4me3) histone marks in the L isoform deficient macrophages. Influenza A virus (IAV) infection, known to stimulate a wide array of anti-viral responses, caused a differential redistribution of PU.1 binding between proximal promoter and distal sites and decoupling from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. These finding suggest that the inflammatory differences seen in SP-R210L-deficient macrophages are a result of transcriptional differences that are mediated by epigenetic changes brought about by differential expression of the SP-R210 isoforms. This provides an avenue to explore how the signaling pathways downstream of the receptor and the ligands can modulate the macrophage inflammatory response.

Keywords: Anti-viral inflammation; Influenza; Macrophage phenotype and function; PU.1 SP-R210 (Myo18A) isoforms.

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Conflict of interest statement

Conflict of Interest

Zissis C. Chroneos is co-founder of Respana Therapeutic, Inc. (http://respana-therapeutics.com/) an early-stage company developing therapeutics targeting SP-R210 isoforms. Dr. Chroneos and The Pennsylvania State University own equity in Respana Therapeutics. These financial interests have been reviewed by the University’s Institutional and Individual Conflict of Interest Committees and are currently being managed by the University.

Figures

Figure 1.
Figure 1.. Differentially Expressed genes are associated with upregulation of Innate Immune Sensing Pathways.
(a) Cells were cultured overnight a 2×105 cells/well and removed using Cell Dissociation Media. Cells were washed, blocked with BD Mouse Fc Block, and stained with fluorescent antibodies against specific cell surface markers. Stained cells were analyzed using a LSR II flow cytometer with compensation and gating analysis performed on FlowJo v 9.9.5. Data plotted is mean of mean fluorescence intensity ± S.E. (n=3), **, adjusted p-value <0.005 compared to WT; ***, adjusted p-value <0.0005 compared to WT. (b, c) RNA isolated from WT and SP-R210L(DN) cells cultured overnight was sequenced and aligned to the mm10 database using hisat2 with read counts obtained using featurecounts. Count data was then compared between genotypes with three replciates per cell type using deseq2. Differently expressed genes between SP-R210L(DN) and WT cells (bwere filtered by p-value<0.05. These gene sets were then filtered using the MGI Immune Genes database to elucidate differentially expressed immune genes (c – SP-R210L(DN) vs WT) The genes were also labeled in red in (b). Differentially expressed RNA genes were mapped to KEGG pathways using the fgsea R package. (d) Upregulated pathways were compared between SP-R210L and WT cells. Enrichment plots for Cytosolic DNA Sensing Pathway and TLR Signaling Pathway were included to show enrichment in genes associated with these gene sets. (e) The enrichment scores for the 10 disease associated pathways with the lowest p-values were plotted for SP-R210L(DN) vs WT cells.
Figure 2.
Figure 2.. PU.1 binding across the genome is altered with SP-R210L depletion.
ChIP was used to precipitate PU.1-bound genomic regions, then sequenced and aligned to the mm10 genome. Concordant peaks between two experimental replicates determined using overlappingpeaks function of ChIPPeakAnno were used for further analysis. (a) Peaks between WT and SP-R210L(DN) cells were compared using ChIPPeakAnno to identify the concordance in peaks between the two genotypes, showing 6140 peaks consistent with both genotypes, and 5365 peaks unique to SP-R210L(DN) cells. The peak distribution across genome was increased in SP-R210L(DN) cells (a). (b) Using Chipseeker, the identified peaks were associated with genomic features for WT and SP-R210L(DN) cells showed decrease PU.1 binding in promoter regions, but with slightly increased binding in 3’ UTR, Exon, and Intron regions. (c) Association between unique ChIP peaks in each cell type and RNA expression. Of the 5365 PU.1 peaks unique to SP-R210L(DN) cells, 437 peaks were associated with RNA transcripts upregulated in WT cells, while 573 peaks were associated with RNA transcripts upregulated in SP-R210L(DN) cells. Of 1011 PU.1 peaks unique to WT cells, 260 peaks were associated with genes with upregulated RNA transcripts in WT cells, and 49 peaks associated with upregulated in SP-R210L(DN) cells. (d) PU.1 associated genes were mapped to Reactome pathways using ReactomePA. Pathway enrichment scores and p-values for WT and SP-R210L(DN) cells were plotted in a heat map; each pathway was associated to larger Reactome pathway families.
Figure 3.
Figure 3.. SP-R210L(DN) cells have altered histone methylation.
(a) ChIP-seq of H3K4me3 was performed for both WT and SP-R210L(DN) cells, showing increased H3k4me3 marks in SP-R210L(DN) cells. (b) Genomic features associated with H3K4me3 marks were analyzed for both cell types, showing decreased H3K4me3 marks in promoter regions, but increased in intron and intergenic regions. This analysis was repeated for H3K9me3 (c, d) and H3K27me3 (e, f) methylation marks. For both H3K9me3 and H3K27me3, there were decreased amounts of these marks in SP-R210L(DN) cells, with similar changes in genomic distribution; there were decreased H3K9me3 and H3K27me3 marks in promoter regions, but increased in Exon, Intron, and Intergenic regions. Using ChIPPeakAnno, it was seen that of the H3K4me marks, only a small proportion is associated with PU.1 peaks in both WT (g) and SP-R210L(DN) (h) cells.
Figure 4.
Figure 4.. PU.1 and H3K4me3 peaks Association with Genes are Altered with SP-R210L(DN) cells.
(a) Bedgraphs were generated for PU.1 and H3K4me3 ChIP and mapped to UCSC mm10 annotated genome. Viewing TLR5 on the UCSC genome browser revealed increased PU.1 and H3K4me3 binding at the promoter region of TLR5 in SP-R210L(DN) cells. Additional bedgraphs of TLR genes can be found in Supplemental Figure 5, Supplemental Figure 6. (b) PU.1 is known to bind its own enhancer region; visualizing PU.1 on the genome browser revealed PU.1 binding sites in the enhancer region of PU.1 in both WT and SP-R210L(DN) cells. (c) Investigating the Myo18A gene revealed several PU.1 peaks in both WT and SP-R210L(DN) cells of varying intensities. H3K4me3 peaks were found at the promoter region of Myo18a, as well as an internal start site. Highlighted in light blue is a predicted PU.1 binding site (UCI Motifmap), with the sequence depicted below. The sequence of a PU.1 peak present internal to Myo18A is also depicted. Within the sequence, a canonical PU.1 binding motif is highlighted in dark green.
Figure 5.
Figure 5.. IAV infection affects PU.1 binding differently in SP-R210L(DN) cells than WT cells.
(a) PU.1 peaks were compared between uninfected and infected WT and SP-R210L(DN) cells to determine which peaks were similar or unique to each condition; infection reduces PU.1 binding in both WT and SP-R210L(DN) cells. (b) IAV infection affects WT and SP-R210L(DN) cells differently; while PU.1 binding with IAV infection has many shared regions with uninfected cells, some unique PU.1 bound regions in WT and SP-R210L(DN) cells were identified with infection. IAV infected WT and SP-R210L(DN) cells showed a majority or PU.1 bound regions to be similar, but each cell type also had numerous peaks that were unique with IAV infection. (c) PU.1 binding was mapped to genomic features for infected and uninfected WT and SP-R210L(DN) cells. Mapping revealed increased distribution of PU.1 binding to promoter regions in WT and SP-R210L(DN) cells, with concomitant decreases in Intron and Intergenic regions. (d) PU.1 associated regions were mapped to Reactome pathways; Pathway enrichment scores and p-values for WT and SP-R210L(DN) cells were plotted in a heat map; each pathway was associated to larger Reactome pathway families.
Figure 6.
Figure 6.. SP-R210L knockdown alters phosphorylation of immune signaling molecules
(a) Clarified cell lysates from WT and SP-R210L(DN) cells infected with PR8 for 3, 6, 12, and 24 hours were probed for phosphorylated and total IRF3 (a), IRF7 (b), NFκB p65 subunit (c, d) and P38 (e). (a) SP-R210L(DN) cells show a trend towards increased baseline IRF3 phosphorylation, but less IRF3 phosphorylation at 24 HPI (n=2). (b) SP-R210L(DN) cells showed increased IRF7 phosphorylation at baseline, with significant increases at 12 and 24 HPI (n=3) (c, d) SP-R210L(DN) cells showed increased Ser276 phosphorylation (c), but decreased Ser536 (d), of NFκB p65 compared with WT at baseline and throughout infection. (n=3) (e) WT cells exhibited increased phosphorylated P38 at 24 hours post infection compared to SP-R210L(DN) cells (n=3). Statistical significance determined by 2-way ANOVA. **, p-value <0.005 comparing between WT and SP-R210L(DN); ***, p-value<0.005 comparing between WT and SP-R210L(DN); +, p-value <0.05 comparing between uninfected and infected time point; ++, p-value <0.005 comparing between uninfected and infected time point; +++, p-value <0.0005 comparing between uninfected and infected time point; ++++, p-value <0.00005 comparing between uninfected and infected time point
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References

    1. Sica A, Mantovani A. 2012. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest 122, 787. - PMC - PubMed
    1. Italiani P, Boraschi D. 2014. From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation. Front Immunol 5, 514. - PMC - PubMed
    1. Stout RD, Jiang C, Matta B, Tietzel I, Watkins SK, Suttles J. 2005. Macrophages sequentially change their functional phenotype in response to changes in microenvironmental influences. J Immunol 175, 342. - PubMed
    1. Hussell T, Bell TJ 2014. Alveolar macrophages: plasticity in a tissue-specific context. Nat Rev Immunol 14, 81. - PubMed
    1. McQuattie-Pimentel AC, Ren Z, Joshi N, Watanabe S, Stoeger T, Chi M, Lu Z, Sichizya L, Aillon RP, Chen CI, Soberanes S, Chen Z, Reyfman PA, Walter JM, Anekalla KR, Davis JM, Helmin KA, Runyan CE, Abdala-Valencia H, Nam K, Meliton AY, Winter DR, Morimoto RI, Mutlu GM, Bharat A, Perlman H, Gottardi CJ, Ridge KM, Chandel NS, Sznajder JI, Balch WE, Singer BD, Misharin AV, Budinger GRS 2021. The lung microenvironment shapes a dysfunctional response of alveolar macrophages in aging. J Clin Invest 131. - PMC - PubMed

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