key: cord-0272634-q831jenz
authors: Luo, Wei-Jia; Yu, Sung-Liang; Chang, Chia-Ching; Chien, Min-Hui; Liao, Keng-Mao; Chang, Ya-Ling; Chuang, Ya-Hui; Chen, Jeremy J.W.; Yang, Pan-Chyr; Su, Kang-Yi
title: HLJ1 amplifies endotoxin–induced sepsis severity by promoting IL-12 heterodimerization in macrophages
date: 2022-01-06
journal: bioRxiv
DOI: 10.1101/2022.01.05.475083
sha: 4cf7961cd1bbc4515bdf2396d7e22ea386522251
doc_id: 272634
cord_uid: q831jenz

Heat shock protein (HSP) 40 has emerged as a key actor in both innate and adaptive immunity, whereas the role of HLJ1, a molecular chaperone in HSP40 family, in modulating endotoxin–induced sepsis severity is still unclear. Here, we use single-cell RNA sequencing to characterize mouse liver nonparenchymal cell populations under LPS (lipopolysaccharide) stimulation, and show that HLJ1 deletion affected IFN-γ-related gene signatures in distinct immune cell clusters. HLJ1 deficiency also leads to reduced serum levels of IL-12 in LPS-treated mice, contributing to dampened production of IFN-γ in natural killer cells but not CD4+ or CD8+ T cells, and subsequently to improved survival rate. Adoptive transfer of HLJ1-deleted macrophages into LPS-treated mice results in reduced IL-12 and IFN-γ levels and protects the mice from IFN-γ–dependent mortality. In the context of molecular mechanisms, HLJ1 is an LPS-inducible protein in macrophages and converts misfolded IL-12p35 homodimers to monomers, which maintains bioactive IL-12p70 heterodimerization and secretion. This study suggests HLJ1 causes IFN-γ–dependent septic lethality by promoting IL-12 heterodimerization, and targeting HLJ1 has therapeutic potential in inflammatory diseases involving activating IL-12/IFN-γ axis.

responses that contribute to various liver diseases in both human and mouse models 64 11 deleted NK cells was comparable to that produced by wild-type NK cells in response 240 to IL-12p70 stimulation ( Figure 4D ). Combined, these results suggested that Hlj1 241 deletion leads to reduced IFN-γ production in not only hepatic but also splenic NK cells, 242

whereas HLJ1 deficiency alters neither the sensitivity to IL-12p70 nor the ability to 243 secrete IFN-γ of NK cells. This suggested that HLJ1 deficiency has little impact on macrophage infiltration before 253 and after LPS administration. Since LPS-induced inflammatory liver injury features 254 activation of the NF-κB pathway and the production of multiple inflammatory 255 cytokines for subsequent IFN-γ activation, we next focused on the cytokines and 256 chemokines generated by hepatic macrophages after LPS stimulation. Transcription 257 levels of proinflammatory cytokines IL-1β, TNF-α, and monocyte chemoattractant 258 protein-1 (MCP-1) did not differ significantly between genotypes, but IL-6 was 259 downregulated in Hlj1 −/− livers compared to Hlj1 +/+ livers ( Figure 5A ). In addition, 260 transcriptional levels of hepatic IL-12, a cytokine contributing to LPS-induced septic 261 death via cytokine storm mediated by the IL-12/18-IFN-γ axis, were lower in Hlj1 −/− 262 than in Hlj1 +/+ mice, although IL-18 expression levels were similar in the two 263 genotypes ( Figure 5B ). Intriguingly, serum levels of IL-12p70 but not IL-6 were 12 dramatically decreased in HLJ1-deficient mice ( Figure 5C ). To investigate the impact 265 of IL-12 on IFN-γ production in the context of HLJ1 deficiency, we administered anti-266 IL-12 neutralizing antibodies intraperitoneally 1 h before the LPS challenge. The serum 267 IL-12 induced by LPS was efficiently neutralized by the antibodies, and serum IFN-γ 268 was also dramatically reduced in both genotypes ( Figure 5D ). It is noteworthy that the 269 serum levels of IFN-γ in anti-IL-12 antibody-injected Hlj1 −/− mice were comparable 270 to those in Hlj1 +/+ mice during sepsis ( Figure 5D ). More importantly, IL-12 271 neutralization significantly reduced the mortality of Hlj1 +/+ mice, which displayed 272 similar survival to Hlj1 −/− mice when challenged with LPS ( Figure 5E ). Combined, 273 these results suggested that HLJ1 is required for LPS-induced IL-12 production, 274 subsequent IFN-γ release, and eventual sepsis death. To confirm that HLJ1 functions exclusively in macrophages, leading to LPS-279 induced IL-12 secretion, IFN-γ-related cytokine storm, and septic death, we performed 280 macrophage transplantation. This was achieved by depleting macrophages and Kupffer 281 cells with clodronate liposomes, followed by adoptive transfer of macrophages from 282 other mice. We intravenously transplanted bone marrow-derived macrophages 283 (BMDMs) into the mice 48 h after the intravenous injection of clodronate liposomes. Figure 6B ). Serum levels of IL-12 and IFN-γ in the LPS-treated 288 mice were significantly reduced as a result of this depletion ( Figure 6C ). After the 289 13 depletion, the LPS-treated Hlj1 −/− mice into which Hlj1 +/+ BMDMs had been 290 transplanted exhibited higher serum levels of IL-12 and IFN-γ than those into which 291 Hlj1 −/− BMDMs had been transplanted ( Figure 6C ). Similarly, macrophage-depleted 292 Hlj1 +/+ mice receiving Hlj1 −/− BMDM transplantation and LPS treatment showed 293 lower serum levels of IL-12 and IFN-γ when compared to with those receiving Hlj1 +/+ 294 BMDMs. Adoptive transfer of Hlj1 +/+ BMDMs into Hlj1 −/− mice led to dramatically 295 elevated mortality rates following an LPS challenge compared to Hlj1 +/+ mice 296 transplanted with Hlj1 +/+ BMDMs ( Figure 6D ). In contrast, the survival of LPS-treated 297 Hlj1 +/+ mice was significantly improved when they were transplanted with Hlj1 −/− 298 BMDMs ( Figure 6D ). Combined, these results indicated that HLJ1 in macrophages is 299 indispensable for maintaining IL-12-mediated IFN-γ production and contributing to 300 septic death in vivo. 301 302 HLJ1 helps IL-12p35 folding and heterodimeric IL-12p70 production 303

Macrophages are major innate immune cells responsible for IL-12 production in 304 response to an endotoxin challenge, which leads to organ dysfunction and even septic 305 shock. We isolated and differentiated BMDMs to investigate the underlying molecular 306 mechanism of HLJ1-modulated IL-12 expression in macrophages. Up to 98% of 307

BMDMs were obtained when differentiated under macrophage colony-stimulating 308 factor (M-CSF) stimulation, demonstrating that the ability to differentiate did not differ and B), indicating that HLJ1 is induced in macrophages rather than in hepatocytes. 337

Since serum levels of HSPs are reported to increase during sepsis, and analysis of 338 survival outcomes of sepsis patients has revealed that increased mortality is associated 339 15 with higher HSP serum levels, we postulated that HLJ1 may be secreted into the blood, 340 and therefore quantified HLJ1 protein in serum from LPS-challenged mice. Indeed, we 341 detected abundant HLJ1 in the serum of Hlj1 +/+ but not Hlj1 −/− mice ( We found HLJ1 to be a modulator of IL-12 protein folding and biosynthesis. 

The HLJ1 knockout (Hlj1 −/− ) mouse was generated by the gene targeting strategy to 518 delete exon2 of Hlj1 gene at the embryonic stage. The syngeneic genetic background 519 of Hlj1 was achieved by backcrossing to C57BL/6 mouse strain over ten generations. 520

Mice deficient in HLJ1 exhibited elevated ER stress-mediated abnormal lipogenesis 521 (paper submitted). All mice were hosted in a pathogen-free facility, maintained in filter-522 

Statistical analysis was performed by using the two-tailed, unpaired Student's t-test 674 with equal variance assumed. Log-rank Mantel-Cox test was used to compare survival 675 curve. Correlation between serum HLJ1 and IL-12 was analyzed by using 676 nonparametric Spearman correlation test. In scRNA-seq, differentially expressed genes 677 of specific cell types were identified by using a Wilcoxon Rank Sum test. Differences 678 were considered statistically significant when p<0.05. 679 680

The raw and processed 10x single-cell sequencing data generated in this study have 

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E3150

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of Streptococcus pneumoniae is involved in bacterial virulence and elicits a 742 strong natural immune reaction via PI3K/JNK

Hepatic response to 745

Meier, S., Bohnacker, S., Klose, C. J., Lopez, A., Choe, C. A., Schmid, P. W. N., . . . respectively. Data presented are means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. 1002Source data 1. Data for graphs depicted in Figure 2D Data presented are means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. 1108Source data 1. Data for graphs depicted in Figure 7A treated with LPS. The HLJ1 induced by LPS helps to reduce the quantity of HMW IL-1137 12p35 homodimers, which becomes IL-12p35 monomers. Properly folded and 1138 assembled IL-12p70, which is composed of IL-12p35 and IL-12p40, is released by 1139 macrophages into the circulation and thereby stimulates NK cells. Eventually, activated 1140 NK cells release IFN-γ in sufficient quantities to lead to death via sepsis.