Phagocytosis by macrophages depends on histamine H2 receptor signaling and scavenger receptor 1

Abstract The histamine H2 receptor (H2R) is a G protein‐coupled receptor that mediates cyclic AMP production, protein kinase A activation, and MAP kinase signaling. In order to explore the multifaceted effects of histamine signaling on immune cells, phagocytosis was evaluated using primary mouse‐derived macrophages. Phagocytosis is initiated by signaling via surface‐bound scavenger receptors and can be regulated by autophagy. Absence of H2R signaling resulted in diminished phagocytosis of live bacteria and synthetic microspheres by primary macrophages from histamine H2 receptor gene (Hrh2)‐deficient mice. Flow cytometry and immunofluorescence microscopy were used to quantify phagocytosis of phylogenetically diverse bacteria as well as microspheres of defined chemical composition. Autophagy and scavenger receptor gene expression were quantified in macrophages after exposure to Escherichia coli. Expression of the autophagy genes, Becn1 and Atg12, was increased in Hrh2 −/− macrophages, indicating upregulation of autophagy pathways. Expression of the Macrophage Scavenger Receptor 1 gene (Msr1) was diminished in Hrh2‐deficient macrophages, supporting the possible importance of histamine signaling in scavenger receptor abundance and macrophage function. Flow cytometry confirmed diminished MSR1 surface abundance in Hrh2 −/− macrophages. These data suggest that H2R signaling is required for effective phagocytosis by regulating the process of autophagy and scavenger receptor MSR1 abundance in macrophages.

in phagocytosis are prone to earlier dissemination of infection, severe sepsis, and subsequently suffer increased mortality (Andrews & Sullivan, 2003;Kim et al., 2017). Reduced phagocytic activity during the first 24 hr after hospital admission is also a known negative predictor for survival in septic patients (Danikas, Karakantza, Theodorou, Sakellaropoulos, & Gogos, 2008). In patients with chronic obstructive pulmonary disease, reduced phagocytosis of common airway pathogens enables bacterial persistence in the lower airways and may contribute to chronic inflammation (Taylor et al., 2010). Given the importance of macrophages in health and disease, elucidating molecular mechanisms that regulate macrophage function and particularly phagocytosis are important for understanding innate immunity.
Phagocytosis is a complex immune process that results in removal and elimination of infectious agents including bacterial cells, misplaced microbes, apoptotic cells, neoplastic cells, or cellular debris. In general, bacterial antigens are recognized via Toll-like receptors (TLRs), pathogen-associated molecular patterns (PAMPs), and NOD-like receptors (NLRs) (Hisamatsu, Ogata, & Hibi, 2008;Rioux et al., 2007;Sheikh & Plevy, 2010). Phagocytosis is commonly initiated by the engagement of surface receptors that trigger plasma membrane and actin cytoskeleton remodeling (Levin et al., 2016(Levin et al., , 2017. This process produces pseudopods that gradually enlarge and engulf the target. Engulfed microparticles enter the phagosome and are subsequently digested. Phagocytosis of microbes differs in terms of relative efficiency among different microbial species, and some organisms may be able to evade or escape killing entirely by professional phagocytes (Perun et al., 2017). Recently, autophagy and phagolysosomal function have also emerged as fundamental elements involved in phagocytosis of invasive microbes (Bonilla et al., 2013;Lima et al., 2011;Rioux et al., 2007). Activation of autophagy has been shown to suppress phagocytosis (Lima et al., 2011;Martinet, Schrijvers, Timmermans, Herman, & De Meyer, 2009), while inhibition or loss of autophagy has been shown to enhance phagocytosis (Bonilla et al., 2013;Song et al., 2017;Zhu, Li, Ding, & Wang, 2018). Importantly, this relationship is mediated by transcriptional control of scavenger receptors by autophagy-related gene products (Bonilla et al., 2013).
Using macrophages derived from histamine receptor-deficient mice, we demonstrated that loss of Hrh2, but not Hrh1, results in decreased phagocytosis efficiency in both bone marrow-derived and peritoneal macrophages. Phagocytosis of phylogenetically diverse bacteria as well as synthetic microspheres is reduced. We determined that Macrophage Scavenger Receptor 1 surface abundance is diminished in Hrh2 −/− bone marrow-derived macrophages which accounts for reduced phagocytosis. Additionally, we demonstrate that expression of key autophagy genes, Atg12 and Becn1, is increased which may be responsible for suppression of scavenger receptor expression.

| Hrh2 is required for phagocytosis by bone marrow-derived macrophages (BMMs)
Hrh1-deficient and Hrh2-deficient mice were generated by CRISPR-Cas9-mediated genome editing. Sequencing confirmed that a 1,546 bp fragment of the Hrh1 gene and a 1,960 bp fragment of the Hrh2 gene were deleted in each mouse ( Figure A1 in Appendix).
Lack of expression was verified by qPCR of isolated macrophages.
These mice are still capable of producing histamine by decarboxylation of L-histidine, and, aside from endogenous histamine produced by mammalian cells, epithelial and immune cells of the intestinal mucosa may be exposed to histamine generated by microbial cells in the intestine (Barcik et al., 2016).
Fluorescence microscopy of macrophages exposed to fluorescent bacteria or microspheres (Figure 1a This phagocytic deficit is also found in cells exposed to Fluoresbrite YG microspheres. Microspheres provide the technical advantages of relatively uniform particle sizes lacking potentially confounding effects of living microbial cells as targets. In Figure 1c Along with E. coli and L. reuteri, murine BMMs were exposed to CFDA-SE-labeled Citrobacter freundii, Salmonella enterica serovar Typhimurium, and Bifidobacterium dentium (representing 4 different bacterial phyla) and phagocytosis was quantified by flow cytometry ( Figure 2). Citrobacter freundii cells were not effectively phagocytosed by WT or histamine receptor-deficient macrophages. Hrh2 −/− BMMs, but not WT BMMs, were defective at phagocytosis of S. enterica serovar Typhimurium and B. dentium. To confirm that endogenous histamine was not playing a role in our model, we assessed BMM supernatants for histamine content by mass spectrometry.
Histamine was not detected in any of the supernatants tested (Table   A1 in Appendix), indicating that our findings regarding phagocytosis were not influenced by endogenous histamine produced by mouse cells. These data demonstrate that H2R is an important modulator of BMM-mediated phagocytosis.

| Hrh2 −/− peritoneal macrophages are deficient in phagocytosis of fluorescent latex microspheres
To study phagocytosis in peripheral macrophages that are more closely associated with the intestinal tract, peritoneal macrophages were isolated from the abdominal cavities of mice. To elicit enough cells for analysis, mice were injected intraperitoneally with Bio-Gel P-100, 4 days prior to macrophage isolation. We did not use thioglycollate in these experiments which is thought to interfere with subsequent phagocytosis analyses (Zhang, Goncalves, & Mosser, 2008). By exposing peritoneal macrophages with Fluoresbrite YG microspheres, we were able to validate decreased phagocytosis in Hrh2 −/− in peritoneal macrophages and enumeration of clearly defined beads per macrophage. Isolated peritoneal macrophages were exposed to Fluoresbrite YG microspheres for 1 hr at a MOI of 10. Fluorescence microscopy of peritoneal macrophages reveals diminished phagocytosis of fluorescent microspheres by Hrh2 −/− macrophages ( Figure 3a). FIJI analysis demonstrates F I G U R E 1 Phagocytosis is reduced in Hrh2 −/− bone marrow-derived macrophages. Phagocytosis of fluorescently labeled bacteria and microspheres was observed in WT, Hrh1 −/− , and Hrh2 −/− bone marrow-derived macrophages. (a) Fluorescence microscopy showed reduced phagocytosis of Fluoresbrite YG microspheres (2 µm) and CFDA-SE-labeled Escherichia coli and Lactobacillus reuteri (green) by Hrh2 −/− macrophages compared to WT or Hrh1 −/− controls. Cells were counterstained with Phalloidin (red), and nuclei were dyed with Hoechst (blue). Bars represent 50 µm, and inset boxes are expanded views of fields of interest. (b and c) Flow cytometry analysis of bone marrowderived macrophages exposed to fluorescent microspheres or CFDA-SE-labeled bacteria confirmed reduced phagocytosis of fluorescent microparticles by Hrh2 −/− bone marrow-derived macrophages. Cells were stained with viability stain, and upon analysis, fluorescence intensity in the FITC channel was measured and compared among populations. Statistical analysis was performed by two-way ANOVA of mean with Bonferroni multiple comparisons. **p < .01, ****p < .0001; n = 3 decreased numbers of microspheres (green foci) per one hundred (100) F4/80-positive peritoneal macrophages ( Figure 3b). The data support our in vitro findings with BMMs, indicating that H2R is a mediator of signaling pathways involved in phagocytosis by both bone marrow-derived and peritoneal (peripheral) macrophages.

| Expression of autophagy genes Becn1 and
Atg12 is increased in Hrh2 −/− macrophages It was previously shown that enhancement of autophagy results in decreased phagocytosis by macrophages (Lima et al., 2011). Therefore, we sought to determine whether enhanced autophagy gene expression could be observed in Hrh2 −/− macrophages. To do this, several key autophagy genes were quantified in bone marrowderived and peritoneal macrophages by qPCR following exposure to E. coli K-12 ( Figure 4). Of the genes analyzed, expression of Becn1 and Atg12, which code for the autophagy pathway components Beclin-1 and Autophagy-related gene 12, was significantly increased in BMMs after 1-hr exposure to E. coli K-12 ( Figure 4). Interestingly, we observed a trend toward greater expression of both genes in untreated Hrh2 −/− BMMs and in peritoneal macrophages with and without exposure to E. coli ( Figure 4). Expression of Atg4, Atg5, and Atg7 genes was not different between WT and histamine receptordeficient macrophages ( Figure A2 in Appendix).

| Macrophage scavenger receptor 1 (MSR1) is reduced in Hrh2 −/− macrophages
Loss of the autophagy-related gene Atg7 in murine macrophages has been demonstrated to increase phagocytosis due to increased scavenger receptor production (Bonilla et al., 2013). To determine whether scavenger receptors were involved in our phenotype, F I G U R E 3 Phagocytosis is reduced in Hrh2 −/− peritoneal macrophages. Phagocytosis of fluorescent microspheres was determined in WT, Hrh1 −/− , and Hrh2 −/− peritoneal macrophages. (a) Fluorescence microscopy was used to analyze phagocytosis in WT, Hrh1 −/− , and Hrh2 −/− peritoneal macrophages at 400x total magnification after 1-hr exposure to Fluoresbrite YG microspheres 2 µm (green). Cells were stained with DAPI (blue) and TRITC-conjugated anti-mouse F4/80 antibody (red). Scale bars represent 50 µm length. Pop-out of enlarged section showing WT macrophages associated with multiple beads. (b) Average number of microspheres associated with macrophages was calculated from counting microspheres and nuclei in 10 separate fields per genotype at 200x total magnification. Statistical analysis was performed by one-way ANOVA of mean. ****p < .0001; n = 3 F I G U R E 2 Hrh2 −/− bone marrow-derived macrophages are deficient in phagocytosis of phylogenetically diverse bacteria. To determine whether the lack of bacterial phagocytosis by Hrh2 −/− macrophages is specific to a certain group of bacteria, WT, Hrh1 −/− , and Hrh2 −/− bone marrow-derived macrophages were exposed to CFDA-SE-labeled Citrobacter freundii, Salmonella enterica ser. Typhimurium, and Bifidobacterium dentium, along with Escherichia coli and Lactobacillus reuteri, all at a MOI of 10. Hrh2 −/− bone marrow-derived macrophages exhibited a deficit in phagocytosis of all bacteria tested compared to WT and Hrh1 −/− bone marrow-derived macrophages, except for C. freundii which was not phagocytosed by WT or Hrh1 −/− macrophages. Statistical analysis was performed by one-way ANOVA of mean for each group. ***p < .001, ****p < .0001; n = 3 we examined expression of key scavenger receptor genes in WT, Hrh1 −/− , and Hrh2 −/− macrophages exposed to E. coli for 1 hr (MOI

| Hrh1 and Hrh2 do not influence TNF production or chemotaxis in bone marrow macrophages
Conflicting evidence exists regarding the role of histamine receptors in macrophage chemotaxis (Czerner et al., 2014;Radermecker et al., 1989). We observed significant changes in phagocytosis in response to histamine receptor status, so we next addressed F I G U R E 4 Expression of autophagy genes Becn1 and Atg12 is increased in Hrh2 −/− macrophages. Expression of autophagy genes was analyzed in bone marrow-derived macrophages and peritoneal macrophages by qPCR. WT, Hrh1 −/− , and Hrh2 −/− macrophages were left untreated or exposed to Escherichia coli K-12 at a MOI of 10 for 1 hr before lysis and RNA isolation. Autophagy gene expression was normalized to Gapdh. Statistical analysis was performed by two-way ANOVA of mean with Bonferroni multiple comparisons. *p < .05, **p < .01, ***p < .001; n = 4 F I G U R E 5 Hrh2 −/− bone marrow-derived macrophages exhibit decreased abundance of Macrophage Scavenger Receptor 1. MSR1 was analyzed by flow cytometry in untreated bone marrow-derived macrophages and those exposed to Escherichia coli for 1 hr. Prior to analysis, macrophages were stained with a viability dye and then stained against the macrophage markers CD11b and F4/80 along with scavenger receptor directed antibodies. (a) Gene expression of Msr1 was quantified in untreated and E. coli-treated bone marrow-derived macrophages by qPCR. Gene expression data were normalized to Gapdh. (b) Median fluorescence intensity was determined in the PE-Vio770 channel, and MSR1 surface abundance was compared. (c) Representative flow cytometry histograms of PE-Vio770 intensity in WT, Hrh1 −/− , Hrh2 −/− populations of bone marrow-derived macrophages demonstrate a leftward peak shift in Hrh2 −/− populations compared to WT and Hrh1 −/− populations. Statistical significance was determined by two-way ANOVA of mean with Bonferroni multiple comparisons. *p < .05, **p < .01, ***p < .001, ****p < .0001; n = 4 whether our macrophages were capable of chemotaxing toward E. coli and responding to E. coli or LPS via cytokine production.
Using fluorescently tagged macrophages and chemotaxis chambers (8 µm pore size), we observed no difference in chemotaxis of our macrophages toward live E. coli ( Figure A3 in Appendix).
Since metabolic stress has been shown to modulate macrophage chemotaxis (Qiao et al., 2009), we examined metabolic activity of our macrophages using the compound resazurin. No differences were found between genotypes of bacterial treatment (E. coli or L. reuteri) ( Figure A3 in Appendix). To determine whether any changes existed in baseline cytokine production, bone marrow macrophages were exposed to 4 hr of E. coli K12 (MOI of 10) or 1 µg/ml LPS and secreted TNF was examined by ELISA ( Figure   A3 in Appendix). Compared to treatment naïve macrophages, addition of E. coli or LPS induced TNF secretion in WT bone marrow macrophages. Interestingly, no differences were observed between Hrh1 −/− and Hrh2 −/− and control WT bone marrow macrophages, indicating that histamine receptor status does not influence TNF production. These data suggest that phagocytosis defects are not due to chemotaxis or the TLR activation in Hrh2 −/− macrophages, but likely due to changes in scavenger receptor abundance. Collectively, these data suggest that Hrh2 influences autophagy pathways and scavenger receptors thereby modulating phagocytosis. Additionally, we demonstrate increased expression of autophagyrelated genes Atg12 and Becn1. Finally, we found that Hrh2 −/− macrophages exhibited diminished gene expression and cell surface abundance of the scavenger protein MSR1. Based on these findings, we conclude that signaling via H2R is involved in regulation of autophagy and scavenger receptor gene expression, thereby influencing macrophage function. Interestingly, we found that defects in phagocytosis were not related to chemotaxis or TLR activation, as Hrh2 −/− BMMs exhibited no defects in chemotaxis toward E. coli or production of TNF. Taken together, we propose the model illustrated in Figure 6 to describe the processes by which H2R is involved in phagocytosis.

| D ISCUSS I ON
The relative capacity for autophagy was determined by quantifying autophagy gene expression in both BMMs and peritoneal macrophages. Becn1 and Atg12 were both significantly increased in expression after exposure to E. coli in Hrh2 −/− BMMs. The data suggest that increased autophagy may result from a loss of H2R signaling. Scavenger receptor transcription has been linked to autophagy proteins previously (Bonilla et al., 2013). The inverse correlation of autophagy and phagocytosis is the result of competition for cellular resources by the two pathways, autophagy and phagocytosis. It has been shown that knocking out a key autophagy gene in mice, Atg7, results in increase phagocytosis by macrophages along with increased scavenger receptor expression (Bonilla et al., 2013). Alternatively, inducing autophagy leads to reduced phagocytosis (Thomas et al., 2012). In our model, we see that decreased phagocytosis in Hrh2 −/− macrophages was likewise accompanied by an increase in expression of two key autophagy genes, Atg12 and Becn1. Formation of both phago-lysosomes and auto-lysosomes requires contribution of membrane material from the plasma membrane; thus, it has been hypothesized that limited cellular membrane resources is the underlying cause of the apparent negative feedback control between autophagy and phagocytosis (Thomas et al., 2012). Bonilla et al. demonstrate the molecular mechanism responsible for the increase in scavenger receptor abundance in Atg7 −/− macrophages. This group demonstrates that increased Marco and Msr1 expression was caused by increased activity of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), the transcription factor for these scavenger receptors.
In autophagy-impaired macrophages, accumulation of p62, which is embedded in the autophagosome, is more available for activation of NFE2L2; thus, Marco and Msr1 expression is increased (Bonilla et al., 2013). Accordingly, in cells with highly active autophagy, a lower abundance of free p62 may result in less activation of NFE2L2 and diminished scavenger receptor transcription. In Hrh2 −/− macrophages, we did not find changes in Atg7 expression, or in expression of Atg4, Atg5, or Atg16 ( Figure A2 in Appendix) F I G U R E 6 Schematic representation of the role of Hrh2 in macrophage-mediated phagocytosis. Histamine H2 receptor signaling is required for appropriate MSR1 production and efficient phagocytosis by suppression of autophagy genes Atg12 and Becn1 in macrophages. Deletion of Hrh2 results in increased Atg12 and Becn1 expression and decreased Macrophage Scavenger Receptor 1 cell surface abundance and reduced phagocytosis which along with Becn1 and Atg12 have been studied in one or more aspects of immunological regulation (Virgin & Levine, 2009).
The data suggest, however, that the mechanistic negative feedback of phagocytosis by autophagy extends to other autophagyrelated gene products as well, as we detected increased Becn1 and Atg12 expression accompanied by decreased Msr1 expression and MSR1 surface abundance.
Scavenger receptors are transmembrane glycoproteins that include CD36, CD68, SR class A, and SR class B receptors, and binding of receptor to their ligands mediates uptake and clearance. The receptors mediate multiple physiologically important cell processes such as the uptake of oxidized lipoproteins as well as recognition and initiation of phagocytosis of microbes by macrophages (Rich et al., 2013). MSR1 is a class A scavenger receptor that is present on macrophages and exhibits broad ligand specificity (Areschoug & Gordon, 2009). Msr1 is upregulated on macrophages in response to exposure to bacteria and results in binding and removal of bacteria by phagocytosis (Arredouani et al., 2005). We examined the expression of key  (Cohen, Bueno de Mesquita, & Mimouni, 2015). Therefore, work should be done to determine whether H2R blocker use in humans, especially immunocompromised individuals, impairs bacterial clearance by reducing macrophage phagocytic ability.

| Histamine receptor-deficient mouse models
Hrh1 and Hrh2 receptor-deficient mouse models on the C57BL/6J background were generated by the Genetically Modified Mouse Core at Baylor College of Medicine using CRISPR-Cas9-mediated deletion of functional gene regions. Mice were bred and housed under specific pathogen-free (SPF) conditions with a 12-hr light cycle, at the Feigin Tower vivarium at Texas Children's Hospital, Houston, TX. For genotyping, tail clippings were digested with proteinase K using DirectPCR Lysis Reagent (Viagen, 101-T) and incubated overnight at 55ºC. All digests were terminated by incubation at 80ºC for 45 min. Crude lysates were used with genotyping primers (Table A2 in Appendix) and NEB Taq DNA polymerase (New England Biolabs, M0270L) for standard PCR analysis. PCR products were run on 1.5% sodium borate agarose gels and visualized using EZ vision3 dye (VWR, N472). Using Primer Set 1, wild-type (WT) mice generate a 530 bp product, while Hrh1-deficient mice generated a 390 bp product. Using Primer Set 2, WT mice generated a 627 bp product while Hrh2-deficient mice generated a 310 bp product. Mice were allocated to experimental groups by genotype, and no randomization or blinding was used for experiments. BMMs and peritoneal macrophages were harvested from these mice after euthanasia.

| Macrophage models
To examine phagocytosis in mouse macrophages, bone marrow-derived macrophages were generated from adult WT and histamine receptor-deficient C57BL/6J mice by culturing bone marrow cells as previously described (Trouplin et al., 2013). Briefly, bone marrow cells were harvested by flushing tibias and femurs, in the presence of 50 nM recombinant murine macrophage colony-stimulating factor (M-CSF) (PeproTech, 315-02) in complete DMEM (DMEM media (ATCC, 30-2002) with heat-inactivated fetal bovine serum (FBS) (Thermo Fisher, 10438026), and 1 × antibiotic/antimycotic (Thermo Fisher, 15240062)) for seven days. Peritoneal macrophages were elicited from adult mice by intraperitoneal injection of 2% Bio-Gel P-100 (Bio-Rad, 1504174), followed by a 4-day rest period as previously described (Ray & Dittel, 2010). After the rest period, macrophages were harvested by peritoneal lavage with 10 ml prewarmed PBS. The resulting peritoneal cell suspension was washed by centrifugation at 600 g for 10 min at 4˚C and then resuspended in complete DMEM and allowed to attach to nontissue culture treated, plastic Petri dishes for 6 hr before washing twice with PBS and detaching with 0.25% Trypsin-EDTA (Thermo Fisher, 25200-056).

| Fluorescence microscopy
For microscopic evaluation, macrophages were stained with trypan blue and counted with the Invitrogen Countess II automated cell counter. The cells were then seeded at a density of 3 × 10 5 live cells per chamber in 4-chamber glass slides (Thermo Fisher, 154526) and allowed to attach for 12 hr. Complete media was replaced with pre- Immunostaining was examined on an upright wide-field epifluorescence Nikon Eclipse 90i (Nikon) with a 10 × ocular lens and the following objectives: 20 × Plan Apo (NA 0.75) differential interference contrast (DIC) objective and a 40 × Plan Apo (NA 0.95) DIC. All images were recorded using a CoolSNAP HQ2 camera (Photometrics) with a SPECTRA X LED light source (Lumencor). FIJI (Fiji Is Just ImageJ) software was used to perform semiquantitative analysis of fluorescent stains by tabulating mean pixel intensity (National Institutes of Health) in ten regions/ per chamber, n = 4-10 mice/group.

| Quantitative real-time PCR
To quantify expression of autophagy and scavenger receptor genes (Becn1,Atg4,Atg5,Atg7,Atg12,and Atg16), RNA was isolated from BMMs and peritoneal macrophages after treatment with E. coli K-12. RNA was isolated from TRIZOL treated cell lysate samples using a column purification kit (QIAGEN, 217004) according to the manufacturer's instructions. Complementary DNA was prepared using the SensiFAST cDNA synthesis kit (Bioline, BIO-65054).
Quantitative PCR analysis was then performed using Fast SYBR Green (Applied Biosystems 4385618) and amplified on the Applied Biosystems QuantStudio3 instrument. Gapdh was used as the house keeping gene, and relative gene expression was analyzed using the comparative Ct method (2-∆∆Ct method). Primers for qPCR analysis are listed in Table A3 in Appendix.

| Liquid chromatography/ mass spectrometry (LC-MS)
To examine endogenous production of histamine by macrophages, increased to 70% B over 5 min; ramp to 80% B for 6 s and held for 1 min; ramp back to 10% B over 6 s and maintained at 10% for a total chromatographic run time of 12 min to re-equilibrate.

| Chemotaxis and viability assays and cytokine quantification by ELISA
For analysis of chemotaxis, macrophages were fluorescently tagged with 10 µM CFDA-SE for 15 min at 37ºC, 5% CO 2 , similar to CFDA-SE staining for bacteria. Fluorescence was confirmed by microscopy.
Cell number and viability was assessed by trypan blue staining using the Countess II as described previously. Cells were resuspended at 4 × 10 5 cells per well in chemotaxis medium (RPMI 1640 supplemented with 0.1% bovine serum albumin (Sigma-Aldrich, A7030).
Macrophages were placed in Corning HTS 8 µm Transwell 96 well permeable supports (Sigma, CLS3374). Chemotaxis medium containing nontagged E. coli K12 (MOI = 10) was added to the bottom wells of the chamber, and the membrane was inserted. The chamber was incubated for 4 hr at 37ºC, 5% CO 2 , and 95% humidity to allow for chemotaxis. Chemotaxis was quantified by measuring the fluorescence of macrophages that had traversed to the bottom chamber (excitation: 485 nm, emission: 528 nm).
To assess the metabolic activity of our macrophages, the resazurin assay was implemented as previously described (Feng & Cohen, 2013). Briefly, the dye resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) (Sigma-Aldrich, R7017) was added at a final concentration of 44 µM, and plates were incubated for 3 hr at 37°C, 5% CO 2 . Cell viability was measured by reading the fluorescence resulting from resazurin reduction to resorufin using a microplate spectrofluorometer at an excitation wavelength of 570 nm and an emission wavelength of 600 nm.
Each condition was run in biological triplicate and in technical duplicate. Prior to ELISA, protein concentrations were measured and normalized for each group according to the manufacturer's instructions (R&D Systems). Colorimetric absorbances were measured spectrophotometrically, and TNF concentrations were calculated from the standard curves according to manufacturer's instructions.

| Statistical analysis
All sample sizes (n-values) indicated in each figure legend correspond to independent biological replicates. Data presented in graphs represent averages and standard deviations. Comparisons between groups were made with either one-or two-way ANOVA (GraphPad v5.04). A p-value of .05 or less was considered significant.

ACK N OWLED G M ENTS
We would like to thank Dr. Chun Shik Park for sharing his expertise

DATA AVA I L A B I L I T Y S TAT E M E N T
All primer sequence data are provided in Tables A2 and A3 in Appendix. F I G U R E A 1 Development of Hrh1-and Hrh2-deficient mouse models. C57B6 mice Hrh1-and Hrh2-knockout mice were generated by CRISPR-Cas9-mediated genome editing of functional exons F I G U R E A 2 Expression of autophagy genes Atg4, Atg5, and Atg7 is not different in histamine receptor-deficient macrophages. Expression of autophagy genes was analyzed in bone marrow-derived macrophages and peritoneal macrophages by qPCR. WT, Hrh1 −/− , and Hrh2 −/− macrophages were left untreated or exposed to Escherichia coli K-12 at a MOI of 10 for 1 hr before lysis and RNA isolation. Autophagy gene expression was normalized to Gapdh. Statistical analysis was performed by two-way ANOVA of mean with Bonferroni multiple comparisons; n = 4 F I G U R E A 3 Histamine receptor status does not affect chemotaxis or TNF production in bone marrow-derived macrophages. Cell viability and key macrophage functions were quantified in WT and histamine receptor-deficient BMMs. (a) Chemotaxis is not different in histamine receptor-deficient macrophages, and analysis of viability (b) shows that exposure to bacteria for 1 hr does not modulate viability in histamine receptor-deficient macrophages. TNF production was quantified in WT and histamine receptor-deficient macrophages after exposure to Escherichia coli for 4 hr (c) or LPS alone for 4 hr (d). Both treatments induced TNF production, but not differently in histamine receptor-deficient macrophages. Statistical significance was determined by two-way ANOVA of mean with Bonferroni multiple comparisons. *p < .05

Robert
F I G U R E A 4 Scavenger receptor gene expression analysis. Expression of scavenger receptor genes was analyzed by qPCR. WT, Hrh1 −/− , and Hrh2 −/− bone marrow-derived macrophages were left untreated or exposed to Escherichia coli K-12 at a MOI of 10 for 1 hr before lysis and RNA isolation. Scavenger receptor gene expression was normalized to Gapdh. Statistical analysis: Two-way ANOVA of mean with Bonferroni multiple comparisons. **p < .001, ****p < .0001; n = 4 F I G U R E A 5 Scavenger receptor protein analysis. Scavenger receptors were analyzed by flow cytometry in untreated bone marrowderived macrophages and those exposed to Escherichia coli for 1 hr. Prior to analysis, macrophages were stained with a viability dye and then stained against the macrophage markers CD11b and F4/80 along with scavenger receptor directed antibodies. Median fluorescence intensity was determined in the PE and PE-Vio770 channels, and protein surface abundance was compared. Statistical significance was determined by two-way ANOVA of mean with Bonferroni multiple comparisons. *p < .01, ****p < .0001; n = 4