Hemojuvelin regulates the innate immune response to peritoneal bacterial infection in mice

ABSTRACT Hereditary hemochromatosis and iron imbalance are associated with susceptibility to bacterial infection; however, the underlying mechanisms are poorly understood. Here, we performed

_in vivo_ bacterial infection screening using several mouse models of hemochromatosis, including _Hfe_ (_Hfe__−/−_), _hemojuvelin_ (_Hjv__−/−_), and macrophage-specific _ferroportin-1_

(_Fpn1__fl/fl_;_LysM-Cre_+) knockout mice. We found that _Hjv_−/− mice, but not _Hfe__−/−_ or _Fpn1__fl/fl_;_LysM-Cre_+ mice, are highly susceptible to peritoneal infection by both

Gram-negative and Gram-positive bacteria. Interestingly, phagocytic cells in the peritoneum of _Hjv_−/− mice have reduced bacterial clearance, IFN-γ secretion, and nitric oxide production;

in contrast, both cell migration and phagocytosis are normal. Expressing _Hjv_ in RAW264.7 cells increased the level of phosphorylated Stat1 and nitric oxide production. Moreover,

macrophage-specific _Hjv_ knockout mice are susceptible to bacterial infection. Finally, we found that Hjv facilitates the secretion of IFN-γ via the IL-12/Jak2/Stat4 signaling pathway.

Together, these findings reveal a novel protective role of Hjv in the early stages of antimicrobial defense. SIMILAR CONTENT BEING VIEWED BY OTHERS EXTRACELLULAR SQSTM1 MEDIATES BACTERIAL

SEPTIC DEATH IN MICE THROUGH INSULIN RECEPTOR SIGNALLING Article 19 October 2020 GLUTAMATE OXALOACETATE TRANSAMINASE 1 IS DISPENSABLE IN MACROPHAGE DIFFERENTIATION AND ANTI-PATHOGEN RESPONSE

Article Open access 05 July 2024 NUPR1 IS A CRITICAL REPRESSOR OF FERROPTOSIS Article Open access 28 January 2021 INTRODUCTION In the 1960s, researchers first suggested a link between iron

metabolism and the immune system [1]. Since then, many iron-related genes have been found to play a role in immune function. For example, the proteins lactoferrin, hepcidin, and Hfe have all

been found to modulate the host defense against bacterial infection [2–6]. Moreover, the expression of several genes involved in iron metabolism, including hemojuvelin (_HJV_) [7], hepcidin

(_HAMP_) [8], and ferroportin1 (_FPN1_) [9, 10], are modulated during inflammation. Interestingly, a recent case report described a patient with hemochromatosis who died from bacterial

infection [11]. These findings prompted us to investigate the putative role of hemochromatosis-related genes in bacterial infection. We therefore screened several hemochromatosis mouse

models for their immune phenotype and susceptibility to bacterial infection. HJV is a bone morphogenetic protein (BMP) co-receptor that regulates the expression of hepcidin [12]. In humans,

mutations in the _HJV_ gene cause juvenile hemochromatosis [13]. In mice, deleting _Hjv_ expression causes a similar iron-overload phenotype [7, 14]. HJV is also a member of the repulsive

guidance molecule (RGM) family [15]. However, the role of HJV in the immune response is currently unknown. Recent studies suggest that two other RGM family members—RGMa and RGMb—play a role

in immune regulation. For example, RGMa is found in bone marrow-derived dendritic cells, where it modulates T cell responses [16]. RGMb is strongly expressed in macrophages, and increased

IL-6 levels were measured in macrophages isolated from _Rgmb_-knockout mice [17]. Notably, the _Hjv_ gene (also known as _RGMc_) is also expressed in macrophages [17], and studies suggest

that lipopolysaccharide (LPS) stimulation induces organ-specific patterns of _Hjv_ expression [7]. However, whether Hjv plays a role in the immune system’s response to bacterial infection is

currently unknown. Here, we used an acute infection mouse model to investigate the potential immunomodulatory roles of hemochromatosis genes. Our results indicate that Hjv plays a major

role in mediating the host innate immune response to bacterial infection. RESULTS _HJV_-KNOCKOUT MICE HAVE INCREASED SUSCEPTIBILITY TO BACTERIAL INFECTION To investigate the function of

hemochromatosis-related genes in response to acute bacterial infection, we administered an intraperitoneal (i.p.) injection of a lethal dose of the Gram-negative bacterium _Salmonella

typhimurium (S. typhimurium) _[5, 18 ] to the following hemochromatosis mouse models: _Hfe_-knockout (_Hfe__−/−_) mice (Figure 1a), hemojuvelin-knockout (_Hjv__−/−_) mice (Figure 1b), and

macrophage-specific ferroportin1-knockout (_Fpn1__fl/fl_;_LysM-Cre_+) mice (Figure 1c). Although all three models are reported to develop systemic iron overload [9], only the _Hjv__−/−_ mice

were highly susceptible to _S. typhimurium_ infection, reaching 100% mortality within 20 h of infection. Two factors may contribute to the high sensitivity of _Hjv__−/−_ mice to bacterial

infection. First, of these three models of hemochromatosis, _Hjv__−/−_ mice develop the most severe form of iron overload; second, _Hjv__−/−_ mice have the lowest basal hepcidin levels. With

respect to the first factor, iron overload has been shown to facilitate bacterial proliferation [19, 20]. Consistent with this finding, the _Hjv__−/−_ mice had significantly more bacteria

in the major organs and peritoneal fluid compared with wild-type mice (Figure 1d). In order to reduce the tissue iron burden in _Hjv__−/−_ mice to wild-type levels, we fed 4-week-old

_Hjv__−/−_ mice an iron-deficient (ID) diet for four weeks producing _Hjv__−/−_-ID mice, which had comparable iron contents compared with wild-type mice (Supplementary Figures S1A and C).

Injecting _Escherichia coli_ into both _Hjv__−/−_ and _Hjv__−/−_-ID mice caused high mortality (Figure 1e). Given its relatively higher safety compared with _S. typhimurium_, we used _E.

coli_ infection for further experiments. Consistent with increased mortality, the peritoneal cavity of _Hjv__−/−_-ID mice contained higher levels of bacterial colony-forming units (CFUs)

(Figure 1f). Thus, decreasing iron levels to wild-type levels was not sufficient to fully restore bacterial clearance in _Hjv__−/−_ mice; therefore, factors other than iron overload likely

contribute to the susceptibility of _Hjv__−/−_ mice to bacterial infection. Hepcidin, the downstream target of Hjv encoded by the _Hamp_ gene, has been reported to regulate inflammatory

responses in mice [4]. Moreover, hepcidin-knockout mice are sensitive to LPS stimulation [4]. Because hepcidin levels are low in _Hjv__−/−_ mice under basal conditions [7], we hypothesized

that low hepcidin levels may contribute to the high susceptibility of _Hjv__−/−_ mice to bacterial infection. To test this hypothesis, we challenged _Hjv__−/−_ mice with LPS and

D-galactosamine [21] to induce endotoxic shock. We found that the mortality rate was similar between LPS-treated _Hjv__−/−_ mice and LPS-treated wild-type mice (Figure 1g). Interestingly,

LPS stimulation induces hepcidin in _Hjv__−/−_ mice [7], and _Hamp_ mRNA levels were similar between wild-type and _Hjv__−/−_ mice following _E. coli_ infection (Supplementary Figure S2A).

Moreover, serum TNF-α levels (measured 1 h after LPS stimulation) and IL-6 levels (measured 2.5 h after LPS stimulation) were similar between _Hjv__−/−_ and wild-type mice (Supplementary

Figure S2B and C). Taken together, these results suggest that reduced hepcidin expression does not contribute significantly to the increased mortality of _Hjv__−/−_ mice in response to

bacterial infection. To determine whether _Hjv__−/−_ mice are also susceptible to infection by Gram-positive bacterium, we gave _Hjv__−/−_ and wild-type mice a lethal i.p. dose of the

Gram-positive bacterium _Staphylococcus aureus (S. aureus)_ and found considerably higher mortality in the infected _Hjv__−/−_ mice (Figure 1h). Thus, our acute lethal infection experiments

indicate that _Hjv__−/−_ mice are highly susceptible to infection by both Gram-negative and Gram-positive bacteria, suggesting that Hjv may play an essential role in the host defense against

bacterial infection. IMPAIRED MICROBIAL CLEARANCE IN _HJV__−/−_ MACROPHAGES AND NEUTROPHILS Our findings suggest that bacterial clearance is impaired in _Hjv__−/−_ mice. However, bacteremia

induced by an intravenous (i.v.) injection of _E. coli_ [22, 23] caused similar mortality between _Hjv__−/−_ and wild-type mice (Figure 2a). Houghton _et al._ [18] reported that impaired

macrophage function within the peritoneal cavity can result in bacterial dissemination to other sites. Thus, local defense processes are partially independent of systemic protection and may

play an essential role in host survival. Based on these findings, we hypothesized that the local defense process is impaired in the peritoneal cavity of _Hjv__−/−_ mice. We therefore

measured the immune response in the peritoneal cavity following bacterial infection. In the early stages of bacterial infection, macrophages and neutrophils mediate bacterial clearance [24].

To determine the relative proportions of these two cell types in infected _Hjv__−/−_ mice, we isolated peritoneal cells, stained the cells with F4/80 and Gr-1, and performed flow cytometry

[25]. Our analysis revealed that 6–12 h after bacterial infection, neutrophils (F4/80−, Gr-1+) and macrophages (F4/80+) comprised ~80% and 10%, respectively, of the total population of

phagocytes; after 24 h, the percentage of neutrophils decreased to ~50%, and the percentage of macrophages increased to ~40% (Figure 2b). Interestingly, the relative proportions of both cell

types were similar between _Hjv__−/−_ and wild-type mice following bacterial infection, indicating that the migration of macrophages and neutrophils to the peritoneal cavity is intact in

_Hjv__−/−_ mice. Next, we examined further the phagocytic function of peritoneal cells. Primary peritoneal macrophages were obtained from _Hjv__−/−_ and wild-type mice by peritoneal lavage

with Thioglycollate medium [26]. We then incubated the cells with Alexa Fluor 488 conjugated heat-killed _E. coli_ and measured phagocytosis using flow cytometry. We found no difference

between _Hjv__−/−_ macrophages and wild-type macrophages with respect to phagocytosis ratio (Figure 2c) or mean fluorescence intensity (Figure 2d), suggesting that _Hjv__−/−_ macrophages

have normal phagocytic activity. Despite normal phagocytosis measured _in vitro_, we observed more GFP fluorescence in peritoneal cells obtained from _Hjv__−/−_ mice 12 h after infection

with GFP-expressing _E. coli_ compared with wild-type mice (Figure 2e). This stronger immune response in _Hjv__−/−_ mice may be due to bacterial overload in the phagocytes; on the other

hand, bacteria in the peritoneal cavity were over-proliferated in _Hjv__−/−_ mice (Figure 1f). Taken together, these data suggest that although more bacteria are engulfed by _Hjv__−/−_

phagocytes, they are not efficiently killed and/or degraded. REDUCED IFN-Γ EXPRESSION IN _HJV__−/−_ NEUTROPHILS AND MACROPHAGES To test for a possible immune deficiency in _Hjv__−/−_ mice,

we measured the levels of circulating inflammatory cytokines, which generally reflect the presence of an ongoing immune response and can be used to monitor bacteria clearance. We therefore

collected serum samples after infecting mice with of _E. coli_ (2×108 CFU) by i.p. injection; serum was collected 1, 2.5, and 6 h after infection for measuring TNF-α, IL-6 and IFN-γ levels,

respectively. Both TNF-α and IL-6 levels were approximately twofold higher in the serum of _Hjv__−/−_ mice compared with wild-type mice; in contrast, IFN-γ levels were significantly lower in

the _Hjv__−/−_ mice (Figure 3a). IFN-γ is secreted primarily by NK (natural killer) cells and T cells [27]. However, in our acute infection models, the mice died within 24 h, well before NK

cells and T cells are activated [27]. We first confirmed that splenocytes isolated from infected _Hjv__−/−_ mice and wild-type mice have extremely low IFN-γ secretion (Supplementary Figure

S3A). We then examined several cell types in the peritoneal cavity. Macrophages and neutrophils are potential sources of IFN-γ in the early stage of infection [28, 29]. Thus, a lack of IFN-γ

autocrine secretion may underlie the impaired killing capacity of _Hjv__−/−_ macrophages and neutrophils. Indeed, the myeloid lineage cell fraction in the peritoneal cavity contained

considerable numbers of IFN-γ positive (IFN-γ+) cells (Figure 3b). Six hours after _E. coli_ infection, 17.5 and 4% of wild-type and _Hjv__−/−_ peritoneal myeloid lineage cells were IFN-γ+

(Figure 3b). Twelve hours after infection, the percentage of IFN-γ+ cells decreased in both genotypes; however, the percentage of IFN-γ+ cell was still 2-fold higher in the wild-type mice

than in the _Hjv__−/−_ mice. We then examined the identity of the IFN-γ+ cells 6, 12, and 24 h after infection. For wild-type peritoneal cells, 6 h after infection, 75% and 25% of the IFN-γ+

cells were neutrophils (that is, Gr-1+, F4/80− cells) and macrophages (that is, F4/80+ cells), respectively; however, 12 h after infection, the percentage of IFN-γ+ neutrophils cells

decreased to only 13%, and the percentage of macrophages increased to 87%. Moreover, in wild-type mice we detected only a very small proportion of T cells (CD8b+) and NK cells (NK1.1+)

within the peritoneal population of IFN-γ+ cells 12 h after infection (Supplementary Figure S3B); thus, phagocytes—rather than NK cells or T cells—are the principal source of IFN-γ in the

early stages of peritoneal infection. Recent studies found that cellular iron content can affect macrophage function [19, 30]. In _Hjv__−/−_ mice, splenic iron content is low (Supplementary

Figure S1A) because ferroportin retained in the plasma membrane exports more iron out of these macrophages in the spleen. To exclude this effect, we generated macrophage-specific

_Hjv_-knockout (_Hjv__fl/fl_;_LysM-Cre_+) mice; iron content in the major organs and macrophages of these mice was normal (Supplementary Figure S1B and D), and _E. coli_-induced mortality

was similar between _Hjv__fl/fl_;_LysM-Cre_+ mice and _Hjv_−/−-ID mice (compare Figure 3c with Figure 1e). Notably, treating _Hjv__fl/fl_;_LysM-Cre_+ mice with IFN-γ (10 units per mouse)

[31, 32 ] significantly improved survival following _E. coli_ infection (Figure 3d). Next, we examined the specific functions of macrophages and neutrophils. We performed adoptive transfer

experiments by injecting 2×106 wild-type neutrophils or macrophages into the peritoneal cavity of _Hjv_−/− mice; 4 h later, these mice were challenged with a lethal dose of _E. coli_ (2×108

CFU). Although transferring wild-type neutrophils conferred no protection to these mice (Figure 3e), transferring wild-type macrophages conferred a significant protective effect against

bacterial infection compared with _Hjv_−/− macrophages (Figure 3f). Thus, impaired macrophage function, possibly due to reduced IFN-γ production and/or secretion, appears to play a key role

in the susceptibility of _Hjv__−/−_ mice to bacterial infection. DECREASED IFN-Γ SECRETION IN _HJV__−/−_ MACROPHAGES REDUCES THEIR NITRIC OXIDE PRODUCTION AND KILLING CAPACITY IFN-γ is a

critical mediator of protective immunity against both viral and bacterial infections; consistent with this role, _IFN-γ_-knockout mice are highly susceptible to bacterial infection [33, 34].

Specifically, peritoneal cells produce IFN-γ and are essential for the survival of infected mice, and bacterial clearance by these cells is impaired when IFN-γ secretion is reduced [35]. To

further investigate the function of IFN-γ release by macrophages in the early stages of bacterial infection, we isolated and cultured peritoneal macrophages, then stimulated the cells with

_E. coli_ that was heat-killed to prevent lethality due to uncontrolled bacterial replication, thereby mimicking infection _in vivo_. Twenty-four hours after stimulation, both _Hjv__−/−_ and

_Hjv__fl/fl_;_LysM-Cre_+ macrophages secreted significantly fewer amounts of IFN-γ compared with wild-type macrophages (Figure 4a); in contrast, both TNF-α and IL-6 were produced at

wild-type levels (Supplementary Figure S4A and B). In macrophages, IFN-γ activates the enzyme iNOS (inducible nitric oxide synthase), thereby increasing nitric oxide (NO) production in these

cells [36, 37]. We used thioglycollate-elicited macrophages as our primary cell model, as NO synthesis is robust in these cells [38, 39]. After cells were stimulated with heat-killed _E.

coli_, both phosphorylated Stat1 (pStat1, an upstream activator of iNOS) and iNOS expression were significantly lower in _Hjv__−/−_ macrophages compared with wild-type cells (Figure 4b and

c). Moreover, the amount of NO released was significantly lower in both _Hjv__−/−_ and _Hjv__fl/fl_;_LysM-Cre_+ macrophages compared with wild-type cells (Figure 4d). Applying exogenous

IFN-γ (100 ng ml−1) to _Hjv__−/−_ macrophages restored both their pStat1 levels and NO secretion (Figure 4e and f). Macrophages utilize NO production to confer protection from invading

pathogens [40]. We, therefore, collected thioglycollate-elicited macrophages from both _Hjv__fl/fl_;_LysM-Cre_+ mice and _Hjv__fl/fl_;_LysM-Cre__−_ control mice. After 90 min in culture, the

bacterial killing capacity of the _Hjv__fl/fl_;_LysM-Cre_+ macrophages was significantly decreased compared with _Hjv__fl/fl_;_LysM-Cre__−_ controls (that is, the _Hjv__fl/fl_;_LysM-Cre_+

macrophages contained significantly more CFUs than the control cells) (Figure 4g). Notably, pretreating both _Hjv__fl/fl_;_LysM-Cre_+ and control macrophages with IFN-γ significantly

increased bacterial killing capacity, thereby rescuing the phenotype in _Hjv__fl/fl_;_LysM-Cre_+ macrophages. Moreover, co-treating cells with IFN-γ and an IFN-γ neutralizing antibody

(NeuAb) significantly reduced the bacterial killing capacity of both _Hjv__fl/fl_;_LysM-Cre_+ and control macrophages (Figure 4g). To investigate other possible mechanisms involved in the

killing capacity of macrophages, we also measured the level of reactive oxygen species (ROS) within the cells. However, we found no significant difference between _Hjv__−/−_ and wild-type

macrophages (Figure 4h). We therefore conclude that impaired IFN-γ production in _Hjv_−/− macrophages plays a principal role in the reduced killing capacity of these cells, possibly

explaining the unrestricted growth of bacteria in the peritoneal cavity and major organs of _Hjv_−/− mice. HJV IS UPREGULATED AND FACILITATES THE NO CASCADE IN INFECTED MACROPHAGES We found

that elicited wild-type macrophages treated with heat-killed _E. coli_ had significantly increased levels of _Hjv_ mRNA (Figure 5a); in contrast, and consistent with a previous report [7],

_Hjv_ expression was decreased in the liver (Supplementary Figure S5). These results suggest that Hjv may play a regulatory role during infection. Interestingly, stimulating

_Hjv_-transfected RAW264.7 cells (a murine macrophage cell line) with either LPS or heat-killed _E. coli_ led to significantly increased NO production compared with control cells and cells

transfected with an empty vector (Figure 5b). Consistent with these results, both iNOS expression (Figure 5c) and pStat1 levels (Figure 5d) were also increased. These results suggest that

Hjv plays a key role in both iNOS expression and NO production in macrophages. Given that IFN-γ treatment rescued iNOS signaling (Figure 4e, f), Hjv likely modulates the iNOS cascade

primarily via IFN-γ signaling. IL-12/STAT4 SIGNALING IS IMPAIRED IN _HJV__−/−_ MACROPHAGES Hjv is a major regulator of Bmp/Smad signaling [41]. However, whether Bmp/Smad signaling is altered

in _Hjv__−/−_ macrophages and/or whether crosstalk exists between the inflammatory response and Bmp/Smad signaling is not currently known. Bmp6 can induce NO production in macrophages

independent of Smad [42], and inflammatory stimuli can reduce Smad1/5/8 phosphorylation in macrophages [43]. However, we found similar levels of phosphorylated Smad1/5/8 levels between

_Hjv__−/−_ and wild-type macrophages following stimulation with heat-killed _E. coli_ (Supplementary Figure S6A). When we treated wild-type macrophages with LDN193189, a classic inhibitor of

Bmp/Smad signaling, [44] pSmad1/5/8 levels were significantly reduced (Supplementary Figure S6A), whereas iNOS expression was increased (Supplementary Figure S6B). Moreover, the effect of

heat-killed _E. coli_ on Bmp/Smad signaling was unaffected by Hjv deletion (Supplementary Figure S6A). Together, these results indicate that Bmp/Smad signaling is likely not altered in

_Hjv__−/−_ macrophages during bacterial infection. Both IL-18 [45] and IL-12 [46] are key regulators of IFN-γ secretion. Therefore, to investigate the possible mechanism by which Hjv

regulates IFN-γ, we collected serum from _Hjv__−/−_ and wild-type mice following _E. coli_ infection. We found no significant difference between _Hjv__−/−_ and wild-type mice with respect to

either IL-12 (Figure 6a) or IL-18 (Figure 6b). We therefore hypothesized that the capacity for IL-12 and IL-18 to induce IFN-γ might be reduced in _Hjv__−/−_ mice. To test this hypothesis,

we examined the signaling pathways downstream of IL-12 and IL-18. Previous studies showed that IL-18 regulates IFN-γ via the MAP kinase (MAPK) pathway, [47] and IL-12 drives the

phosphorylation of Stat4 to induce IFN-γ expression. [48] To investigate the role of these two pathways, we measured phosphorylated MAPK proteins and phosphorylated Stat4 in macrophages

stimulated with heat-killed _E. coli_. We found no defect in _Hjv__−/−_ macrophages with respect to p38 (Figure 4c), Erk1/2, or Jnk phosphorylation (Figure 6c). However, _E. coli_ stimulated

phosphorylation of Stat4 was significantly impaired in _Hjv__−/−_ macrophages compared with wild-type macrophages (Figure 6d). Consistent with reduced Stat4 phosphorylation, Jak2

phosphorylation was also reduced in _Hjv__−/−_ macrophages compared with wild-type cells (Figure 6e). Finally, we performed _in vitro_ co-immunoprecipitation experiments using HEK293 cells

co-transfected with Flag-tagged HJV and Myc-tagged IL-12RB1 (the beta-1 subunit of the IL-12 receptor). Although weakly pulled down by IL-12RB1-Myc, HJV-Flag was highly effective at pulling

down IL-12RB1-Myc (Figure 6f), suggesting that HJV may play a direct role in IL-12 signaling. Taken together, these results suggest that HJV may regulate IFN-γ in macrophages via the

IL-12/Jak2/Stat4 pathway. DISCUSSION A clear relationship between hemochromatosis and infection has been established by several groups [49–52]. The majority of these results suggest that

both HFE and Hepcidin (encoded by the _HFE_ and _HAMP_ genes, respectively) play a protective role against bacterial infection in human and mouse. Here, we performed a novel screen using

several hemochromatosis mouse models to investigate the mechanisms that underlie the putative link between iron metabolism and immunity. Importantly, our _in vivo_ screening strategy

differed from previous studies [5, 18] in that we focused on the acute infection phase. Thus, our bacterial dose was based on the lethal dose established for modeling severe peritonitis in

129/svj mice [18]. Of the three hemochromatosis mouse models in our initial screen, we found that only _Hjv__−/−_ mice were highly susceptible to bacterial infection, and we excluded iron

burden as the main contributing factor using iron-deficient _Hjv__−/−_ (_Hjv__−/−_-ID) mice. However, under standard dietary conditions, splenic iron content is lower in _Hjv__−/−_ mice than

in wild-type mice [7], and placing _Hjv__−/−_ mice on an iron-deficient diet lowered iron content in the macrophages even further (Supplementary Figure S1A). These factors complicate the

phenotype of _Hjv__−/−_ mice and may have affected survival. Therefore, after we identified which specific immune cells (that is, macrophages and neutrophils) are functionally impaired in

_Hjv__−/−_ mice, we performed survival experiments using _Hjv__fl/fl_;_LysM-Cre_+ mice, in which Hjv expression is deleted selectively in macrophages and neutrophils. Our results revealed

that _Hjv__fl/fl_;_LysM-Cre_+ mice have a survival profile similar to _Hjv__−/−_-ID mice. Given that the iron content in the major organs and macrophages of _Hjv__fl/fl_;_LysM-Cre_+ mice was

similar to wild-type littermates (Supplementary Figure S1B and C), we conclude that iron burden does not play a major role in the high susceptibility of _Hjv__−/−_ mice to bacterial

infection. We observed significantly reduced levels of circulating IFN-γ in _Hjv__−/−_ mice 6 h after bacterial infection. IFN-γ is primarily secreted by T helper type 1 (Th1) cells [53] and

NK cells [54]. However, given the time frame of the increased IFN-γ levels (that is, within 6 h), the principal source of IFN-γ during the early stages of infection is likely not Th1 or NK

cells. Interestingly, peritoneal macrophages and neutrophils have been reported as a source of IFN-γ [55–58], although this remains controversial [59]; nevertheless, it is worth noting that

Schleicher _et al._ [59] treated peritoneal cells with IL-12, IL-18, and LPS for 72 h, which differs from our bacterial infection model (6–24 h). A recent study in mice also found that Gr-1+

cells produce IFN-γ in the early stages of _Streptococcus_ infection, and these cells are responsible for promoting the survival of infected mice [35]. We therefore conclude that peritoneal

phagocytes secrete IFN-γ, thereby self-activating via an autocrine pathway, providing an essential step in fighting peritoneal infection and improving survival. Our results indicate that

_Hjv_ deletion affects the IL-12/Jak2/Stat4 signaling pathway, but not the IL-18/MAPK pathway. Nevertheless, additional research is warranted in order to better understand the underlying

mechanisms. In conclusion, we identified a novel role for HJV in mediating bacterial clearance via macrophages. Our findings indicate that HJV may serve as a potential target for boosting

protection against bacterial peritoneal infection, thereby improving clinical outcome. MATERIALS AND METHODS MICE _Hfe__−/−__, Hjv__−/−__, Fpn1__floxflox_, _Hjv__flox/flox_ and wild-type

mice were maintained on the 129S6/SvEvTac background and were housed in a specific pathogen-free animal facility [9, 14, 60, 61]. _Fpn1_ knockout in macrophages (_Fpn1__fl/fl_;_LysM-Cre_+)

was achieved by crossing _LysM_-_Cre_ mice [62] with _Fpn1__floxflox_ mice, and _Hjv_ knockout in macrophages (_Hjv__fl/fl_;_LysM-Cre_+) was achieved by crossing _LysM-Cre_ mice [62] with

_Hjv__flox/flox_ mice. LysM-Cre mice were maintained as heterozygotes. All animal experiments were approved and performed in accordance with guidelines established by the Institutional

Animal Care and Use Committee of the School of Medicine, Zhejiang University. _IN VIVO_ INFECTION MODELS AND BACTERIAL GROWTH MEASUREMENTS Mice were infected with various bacterial species

via i.p. or i.v. injection in order to induce peritonitis and bacteremia, respectively. _S. typhimurium_, _E. coli_ and _S. aureus_ were grown in LB and isolated on LB agar plates. For the

IFN-γ rescue experiments, 10 units of IFN-γ in saline (or saline alone) were subcutaneously injected into the right flank of the mice, immediately followed by an i.p. injection of _E. coli_

TOP10 bacteria, which carries the GFP gene on the Puc19 plasmid [63]. Organ/peritoneal cavity colony-forming units (CFUs) were determined by plating dilutions of organ homogenates or

peritoneal lavage fluid on LB plates. DIETARY INDUCTION OF IRON DEFICIENCY To induce iron deficiency, 4-week-old mice were fed a diet containing reduced iron content (0.9 mg Fe kg−1,

AIN-76A-diet, D08080402, Research Diets, Inc., New Brunswick, NJ, USA) for 4 weeks. Standard diet contained 50 mg Fe kg−1, (AIN-76A-diet, D08080401, Research Diets, Inc.). PHAGOCYTOSIS ASSAY

AND MACROPHAGE KILLING ASSAYS For the phagocytosis assay, thioglycollate-stimulated peritoneal macrophages were incubated with Alexa Fluor 488-conjugated heat-killed _E. coli_ (Molecular

Probes) for 1 h. Extracellular fluorescence was then quenched by adding 100 μl of trypan blue [64]. Cells were then washed in cold PBS and examined using fluorescence microscopy or flow

cytometry (FACS analysis). For the killing assay, thioglycollate-stimulated peritoneal macrophages were incubated with TOP10 _E. coli_ for 20 min. The macrophages were then washed with

phosphate-buffered saline (PBS) to remove the bacteria, incubated in 100 μg ml−1 gentamycin in DMEM for 20 min (to kill extracellular bacteria), and then incubated for an additional 120 min

in 25 μg ml−1 gentamycin in DMEM. CFUs were then measured from macrophage lysates [40, 65]. In some experiments, the cells were pretreated for 12 h with IFN-γ (Peprotech) in the presence or

absence of IFN-γ-neutralizing antibody (NeuAb; bp0055, Bio X Cell) prior to adding the bacteria. ELISA ELISA kits for measuring IL-6, TNF-α and IFN-γ were purchased from R&D Systems.

ELISA kits for measuring IL-12 and IL-18 were purchased from Pierce. Sera were collected from _E. coli_-infected wild-type and _Hjv__−/−_ mice at indicated time. Briefly, 50 μl serum, blank

or standard were added in 96-well plate, incubated at room temperature for 2 h, washed by wash buffer for five times. Then, 100 μl conjugated antibodies were incubated at room temperature

for 2 h, washed by wash buffer for five times. TMB mix (100 μl) was added into wells for 30 min, and stop solution was then added to stop the reaction. OD values were read at 450 nm by

Microplate reader (Molecular Devices, Sunnyvale, CA , USA). ANTIBODIES Primary antibodies: rabbit anti-pStat1 (1:1000, Cell Signaling Technology [CST, Boston, MA, USA]), rabbit anti-Stat1

(1:1000, CST), rabbit anti-pErk1/2 (1:1000, CST), rabbit anti-Erk1/2 (1:1000, CST), mouse anti-pJnk (1:1000, CST), rabbit anti-Jnk (1:1000, CST), rabbit anti-pStat4 (1:500, Santa Cruz (Santa

Cruz, CA, USA)), rabbit anti-Stat4 (1:1000, CST), rabbit anti-pJak2 (1:500, CST), rabbit anti-Jak2 (1:1000, CST), rabbit anti-pSmad1/5/8 (1:1000, CST), rabbit anti-Smad1 (1:1000, CST),

rabbit anti-pSmad3 (1:1000, CST), rabbit anti-Smad3 (1:1000, CST), rabbit anti-FLAG (1:1000, CST), mouse anti-Myc (1:1000, CST), or mouse anti-β-actin (Sigma, San francisco, CA, USA).

Secondary antibodies: peroxidase-conjugated goat anti-Rabbit or Goat anti mouse IgG (Proteintech (Chicago, IL,USA), 1:2000). WESTERN BLOT ANALYSIS Western blot was perform as previously

described [9]. Briefly, cells/tissues were lysed by RIPA buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail (Sigma) for 10 min; Sonicate for 10 s to complete cell lysis and

shear DNA followed by adding loading buffer (containing 5% β-Me). Then samples were heated to 95–100 °C for 5 min (alternatively 37 °C 30 min for membrane protein detection), cool on ice and

microcentrifuge for 5 min. Samples were loaded onto SDS-PAGE gel and then electrotransfer to PVDF membrane (Bio-Rad, Hercules, CA, USA). The transferred membrane was incubated in blocking

buffer (5% skim milk in TBST) for 1 h followed by incubation with primary antibody overnight at 4 °C. Membrane was washed three times for 10 min each with TBST, and incubated with HRP-linked

secondary antibody for 1 h at room temperature. Wash three times for 10 min each with TBST. After incubation with ECL (Pierce (Rockford, IL, USA), Thermo (Waltham, MA, USA)) mixture for 5 

min, wrap the membrane in plastic and expose to X-ray film. Co-IP experiments were performed using anti-Flag (1:50, CST) and anti-Myc (1:1000, CST) antibodies. REAL-TIME QUANTITATIVE PCR

Total RNA was isolated from peritoneal macrophages or liver tissue using Trizol (Invitrogen, Carlsbad, CA, USA). Real-time PCR was then performed using two-step quantitative real-time PCR

with the primer pairs listed in Supplementary Table S1. The expression level of each target gene was normalized to the corresponding _β-actin_ mRNA level. EXPRESSION OF _HJV_ IN RAW264.7

MACROPHAGES _Hjv_-expressing constructs were cloned into the pCMV-3Tag-3A vector and transfected into RAW264.7 cells using the FuGENE HD transfection reagent (Promega, Madison, WI, USA).

QUANTIFICATION OF NITRIC OXIDE (NO) AND ROS NO concentration was measured using the Griess assay [66]. 50 μl cell medium, blank or standard control were added into a 96-well plate, followed

by adding 50 μl Griess reagent I and II, incubated at room temperature for 10 min, OD values were read at 540 nm by Microplate reader (Molecular Devices). Intracellular reactive oxygen

species were measured using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method [67]. HK-_E. coli_-stimulated macrophages in 6-well plate were incubated in serum-free DMEM-containing

10 μM DCFH-DA for 20 min at 37 °C in cell culture hood. Cells were then detached by trypsin digestion and followed by FACS analysis at the excitation wavelength of 488 nm and emission

wavelength of 525 nm. STATISTICAL ANALYSIS All summary data are presented as the mean±s.e.m. The log-rank test or Mann–Whitney _U_-test was used to analyze the Kaplan–Meier survival curves.

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2012; 366: 277–285. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to Dr Nancy Andrews for generously providing the _Hfe__−/−__, Hjv__−/−__,

Hjv__flox/flox_, and _Fpn1__flox/flox_ mice. We also thank Dr Baoxue Ge at Tongji University for providing the _E. coli_ TOP10 strain carrying the _GFP_ gene on the Puc-19 plasmid. This work

was supported by grants from the National Natural Science Foundation of China (31530034, 31330036, and 31225013 to FW; 31570791 and 91542205 to JM), and Zhejiang Provincial Natural Science

Foundation of China (LZ15H160002 to JM). We thank members of the Wang and Min laboratories for discussion. AUTHOR INFORMATION Author notes * Qian Wu and Yuanyuan Shen: These authors

contributed equally to this work. * Fudi Wang Tel: +86 571 88206385; Fax: +86 571 88206385 E-mail: [email protected] * Fudi [email protected] AUTHORS AND AFFILIATIONS * The First

Affiliated Hospital, Institute of Translational Medicine, School of Public Health, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine,

Zhejiang University, Hangzhou, China Qian Wu, Yuanyuan Shen, Yunlong Tao, Jiayu Wei, Hao Wang, Peng An, Zhuzhen Zhang, Hong Gao, Tianhua Zhou, Fudi Wang & Junxia Min * Department of

Nutrition, Precision Nutrition Innovation Center, School of Public Health, Zhengzhou University, Zhengzhou, China Qian Wu, Hao Wang & Fudi Wang Authors * Qian Wu View author publications

You can also search for this author inPubMed Google Scholar * Yuanyuan Shen View author publications You can also search for this author inPubMed Google Scholar * Yunlong Tao View author

publications You can also search for this author inPubMed Google Scholar * Jiayu Wei View author publications You can also search for this author inPubMed Google Scholar * Hao Wang View

author publications You can also search for this author inPubMed Google Scholar * Peng An View author publications You can also search for this author inPubMed Google Scholar * Zhuzhen Zhang

View author publications You can also search for this author inPubMed Google Scholar * Hong Gao View author publications You can also search for this author inPubMed Google Scholar *

Tianhua Zhou View author publications You can also search for this author inPubMed Google Scholar * Fudi Wang View author publications You can also search for this author inPubMed Google

Scholar * Junxia Min View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS QW, YS, FW and JM designed the experiments. QW, YS, YT, PA, HW, ZZ, JW

and HG performed the experiments. QW, YS, YT, FW and JM analyzed the data. QW, YS, TZ, FW and JM drafted and revised the manuscript. CORRESPONDING AUTHORS Correspondence to Fudi Wang or

Junxia Min. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION ( SUPPLEMENTARY INFORMATION is linked to the online version of the

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http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wu, Q., Shen, Y., Tao, Y. _et al._ Hemojuvelin regulates the innate immune response

to peritoneal bacterial infection in mice. _Cell Discov_ 3, 17028 (2017). https://doi.org/10.1038/celldisc.2017.28 Download citation * Received: 10 May 2017 * Accepted: 13 July 2017 *

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