Table 2.

Summary of discussed comparative studies of HZ/host interactions

Reference	HZ preparation method	Concentration	Reason for concentration	Cell/model	Major findings relevant to this review
[13]	nHZ: In Table 1	115 μg nHZ/well Corresponding to 30 nmol heme/well 50,000 monocytes/well	Not specified	Human PBMC	- Monocytes that phagocytosed nHZ initially exhibited prolonged oxidative burst and then impaired PMA-elicited oxidative burst - Stimulation with iron-stripped nHZ exhibited PMA-elicited oxidative burst, suggesting iron may be responsible for impairment
[15]	nHZ: In Table 1	100 fmol heme/monocyte Corresponding to 50 RBC/monocyte	Not specified	Human PBMC	nHZ induced production of esterified monohydroxy derivatives of polyenoic acids in a complex isomeric pattern, suggesting nonenzymatic oxidation
[17]	sHZ: [43] nHZ: In Table 1	10–50 μg/mL	Not specified	Murine MΦ cell line B10R	nHZ and sHZ alone did not lead to NO generation, while the presence of IFN-γ led to inducible nitrate oxide synthase and NO production
[16]	sHZ: [43] nHZ: In Table 1	10–75 μg/mL	Specified	Murine MΦ cell line B10R	nHZ and sHZ induced ROS generation, predominantly superoxide anion, and chemokines
[9]	sHZ: [43] nHZ: In Table 1	In vitro: 30 or 100 μM In vivo: 1500 μg HZ	Specified	Murine Flt3 ligand-induced BMDCs; Wild-type (WT) C57BL/6 or mutant mice (MyD88−/−, TRIF−/−, TLR2−/−, TLR4−/−, TLR7−/−, and TLR9−/−)	- nHZ increased levels of chemokines and cytokines, while MyD88−/− and TLR9−/− DCs showed decreased production - WT mice injected with nHZ or sHZ exhibited increased MCP-1 and IL-6, while no increases were seen with MyD88−/− and TLR9−/− mice
[8]	sHZ: [43] nHZ: In Table 1	50 ^M nHZ 150 ^M sHZ	Not specified	Murine Flt3 ligand-induced BMDC	- Crude bovine sHZ but not pure sHZ induced TNF-α secretion - DNase and nuclease treatment abolished previously observed immune effects, suggesting DNA, not HZ, mediated effects
[18]	DV: In Table 1	5 ×109 DVs	Not specified	Male Sprague-Dawley outbred rats	- DV membrane, but not merozoites or nHZ, supported the alternative complement pathway - Injected DVs induced complement consumption in vivo and accumulated within splenic phagocytes and within phagocytes in the vasculature of the lung
[19]	DV: In Table 1	Phagocytosis: 3–5 DVs/neutrophil ROS generation and bacterial killing assay. 2–3 DVs/5 neutrophils	Not specified	Healthy human neutrophils, in some experiments + Staphylococcus aureus	- Complement opsonized DVs, but not iRBCs, merozoites, or free nHZ, were readily phagocytosed by neutrophils and rapidly drove oxidative burst - Subsequent responses to S. aureus were blunted with reduced oxidative burst and bacterial killing
[20]	sHZ: [12] nHZ: In Table 1	100 fmol heme/monocyte Corresponding to 50 RBC/monocyte	Not specified	Human PBMC	- nHZ and sHZ bound to fibrinogen - Fibrinogen-HZ complexes interacted with TLR4 and CD11b/CD18, leading to ROS production
[23]	sHZ: [12] nHZ: In Table 1	10 μg/mL	Specified	Primary human ST	nHZ, but not sHZ, increased ERK1/2 phosphorylation, chemokine, and cytokine secretion, increased ICAM-1 expression, and monocyte migration
[26]	nHZ: In Table 1	25–100 μM	Specified	WT C57/B6 BDMCs	- Lower concentrations of HZ in the presence of IFN-γ led to NO production, while higher concentrations led to dose-dependent decrease - Decreased NO production was reversed by replenishing culture medium or supplementing with L-arginine
[61]	sHZ: [12, 75]	0.5, 0.8, 2.5, 4.0, 10, 12.5 and 20 nmol/mL	Specified	Human fetal neurons and astrocytes	Astrocytes and neurons phagocytosed nHZ and exhibited signs of apoptosis
[62]	sHZ: purchased from InvivoGen	200 and 400 μg/mL	Not specified	BV-2 murine microglial cell line ReNcell VM cell line	sHZ caused significant increases in proinflammatory cytokines, caspase activity, NF-κB and NLRP3 pathway activity, microglial-induced neurotoxicity, and direct neurotoxicity
[29•]	DV: In Table 1	Not specified	Not specified	Human neutrophils	NETosis is stimulated by heme but not DVs
[10••]	sHZ purchased from InvivoGen	ROS: 100 μg/ml DC-T cell coculture assay: 100, 50, 20 μg/ml	Not specified	CLEC12A−/− and WT C57/B6 BDMCs; CLEC12A−/− and WT C57L/B6 mice; CD8+ T cells from OT-I transgenic mice	- sHZ caused CLEC12A−/− BMDCs to produce less ROS, suggesting CLEC12A has a role in signaling for ROS production - Culturing CD8+ T cells with CLEC12A−/− BMDCs stimulated with sHZ abrogated effector functions - CLEC12A−/− mice that developed CM had higher survival rates
[77]	sHZ purchased from InvivoGen	50 and 100 ug/mL	Not specified	Human neutrophils	- sHZ was readily phagocytosed by neutrophils and induced intracellular morphological changes, but NETosis was not induced - Fibrinogen impeded sHZ uptake whereas platelets facilitated phagocytosis
[30••]	nHZ: In Table 1	In vitro: P. yoelii nHZ, 30 ug/mL In vivo: P. yoelii nHZ, 1 mg, P. falciparum nHZ, 1–1.5 mg	Not specified	In vitro: Murine splenic phagocytes + L. monocytogenes In vivo: Mice treated with Py nHZ then orally challenged with L. monocytogenes after 7 days; mice treated with Pf nHZ then intranasally challenged with S. pneumoniae after 7 days	- Py nHZ impaired phagocytic clearance of L. monocytogenes - As with natural infection, Py and Pf nHz impaired splenic clearance of L. monocytogenes but not S. pneumoniae - Effect of nHZ was independent of DNA but dependent on nHZ-associated proteins and/or lipids