The following mice were obtained from Jackson Laboratories: C57Bl/6 (stock #000664), CD45.1(#002014), gp91phox ^-/- and ^-/y (#002365), TNFR1^-/- (#003242) and bred in-house in a specific pathogen-free facility at National Jewish Health. Both male and female mice were used at 8-12 weeks of age, in accordance with protocols approved by the IACUC.

Experimental mice were treated with 200μg zymosan in PBS (Sigma) via intraperitoneal (i.p.) injection. Mice were euthanized by CO₂ asphyxiation 20 hours post-zymosan injection for ex vivo culture or 72 hours post-zymosan for MoMac phenotyping. TNFα neutralizing antibody (clone XT3.11, BioXCell) or isotype-matched control antibody was delivered by i.p. injection of 300μg per mouse at 20h and 44h post-zymosan. Peritoneal lavage was performed using 8mL lavage buffer (HBSS/10mM HEPES/1mM EDTA).

Single cell suspensions were made by passing lavage through a 100μm nylon cell strainer. Cells were washed at least once in RPMI culture media (RPMI/10%HI-FBS/100U/mL Penicillin/100U/mL Streptomycin/0.29mg/mL L-glutamine), counted and plated at a constant density of 1x10⁶ total cells/well in 24 well tissue culture treated plates, unless otherwise noted. Neutralizing antibodies or isotype-matched control antibodies ( Table 1 ) were added at the time of plating. Some cells were labeled with 10μM Pacific Blue Succinimidyl Ester (PBSE) before culture. Plates were incubated at 37°C with 5% CO₂.

Conditioned media was collected after 24 hours in culture and centrifuged to pellet cells prior to treatment of newly plated cells. Proteinase K or DNase (Sigma) were used at 50μg/mL for 25 minutes at 37°C followed by heat inactivation of the proteinase by boiling at 95°C for 5 minutes.

To harvest cells for phenotyping, suspension cells were collected into tubes followed by trypsin treatment of adherent cells at 37°C for 5 minutes. Adherent and suspension cells were pooled for analysis.

Cell-free culture supernatants or low volume peritoneal lavage fluid was collected at the indicated timepoints and stored at -80°C with protease inhibitor cocktail (Thermo). Cytokine detection and quantitation was performed using the LEGENDPlex assay (BioLegend) according to the manufacturer’s instructions.

For neutrophil isolation CD45.1 mice were anesthetized using an isoflurane vaporizer and 1μg recombinant mouse CXCL1 (Peprotech) was delivered intranasally in PBS. Bronchoalveolar lavage (BAL) was collected after 3 hours, at which time >97% of recovered leukocytes were neutrophils. BAL cells were labeled with 5μM Carboxyfluorescein succinimidyl ester. After overnight culture ~60% of neutrophils were apoptotic as defined by annexin V staining, performed according to manufacturer’s instructions (BD Bioscience). These were co-incubated for 1 hour with peritoneal lavage cells after 47h in culture at an estimated ratio of 1:1 apoptotic neutrophils: MoMacs before collection for flow cytometry. Engulfing MoMacs were defined as CD64⁺ CFSE⁺ CD45.1^- Ly6G^- to exclude MoMacs that had bound but not internalized target cells.

Cells from lavage or ex vivo culture were resuspended in Flow cytometry buffer (HBSS/3% FBS) with anti-CD16/32 antibody to block Fc receptors. Fluorescently-conjugated staining antibodies were added (detailed in Table 2 ) for >30 minutes. Sytox Blue dye (Invitrogen) was added prior to data acquisition to exclude dead cells. For intracellular cytokine staining, plated cells were pre-treated with 5μg/mL Brefeldin A (BioLegend) +/- 100ng/mL LPS re-stimulation 4 hours prior to collection. Cells were stained for surface markers as described then fixed, permeabilized and stained with anti-cytokine antibody using the FoxP3 staining buffer set (eBioscience, Thermo).

Flow cytometry data was acquired on an LSRII or LSR Fortessa flow cytometer (BD Bioscience) and analyzed using FlowJo v10 (BD Bioscience). tSNE analysis was performed using FlowJo default settings on concatenated fcs files after gating to select live, single MoMacs representing >2 replicate wells/mice per condition after subsampling to equalize numbers of input MoMacs between samples. “Mature” phenotype was defined as that represented by the majority of untreated WT control MoMacs, as described in individual figure legends.

Statistical analysis was performed using Prism software (Graphpad). Details of the statistical tests performed are described in figure legends.

To study MoMac maturation in a controlled environment and identify factors within the CGD milieu that prevent maturation, we developed an ex vivo culture system and method to define maturational state by flow cytometry. Total peritoneal lavage cells were collected from WT and CGD mice 20 hours after the onset of zymosan-induced peritonitis and cultured over time ( Figure 1A ). At this timepoint, MoMacs of both genotypes were relatively immature, and phenotypically and transcriptionally similar (17). The composition of cell types placed in culture differed somewhat between genotypes reflecting their distributions in the peritoneal cavity at the time of harvest: a higher proportion of neutrophils was present in the lavage of CGD mice, and a higher percentage of B lymphocytes present in WT mice ( Supplementary Figure 1 ). Overall cell numbers declined over time, mostly representing the loss of neutrophils within the first 24 hours after plating, while MoMac numbers were similar between genotypes and declined only slightly over time ( Supplementary Figure 1 ). Notably, very few resident peritoneal macrophages were present in lavage at this timepoint for either genotype (Tim4⁺, < 2% of total CD64⁺ cells (19, 20)). The vast majority (>98%) of plated macrophages were Ly6C⁺ MoMacs derived from recruited monocytes in response to zymosan stimulation. In all further analyses MoMacs were identified in peritoneal lavage as live, single cells lacking expression of Ly6G, Siglec F and B220 and expressing CD64 and F4/80.

MoMac maturation over time in culture was investigated via expression of a panel of monocyte/macrophage marker proteins described in the literature. Ly6C is used ubiquitously to describe a population of circulating monocytes in the mouse. Although its molecular function is still unclear, down-regulation of this marker correlates with macrophage maturation (21–23). At 20h post-zymosan, MoMacs from both WT and CGD mice express Ly6C and downregulation was observed over time in both genotypes, although significantly delayed in CGD compared to WT ( Figure 1B ). F4/80 and CD64 are widely used to identify macrophages and were both up-regulated on macrophages compared to circulating monocytes (24). This was observed in our ex vivo cultured WT MoMacs but to a lesser extent in CGD MoMacs over time. CD54 (ICAM1) and the costimulatory molecule CD80, markers of activated pro-inflammatory cells (25), were both up-regulated by CGD MoMacs over time in culture ( Figure 1B ; Supplementary Figure 2 ). In contrast pro-resolving macrophages up-regulate expression of CD36 and CD206, scavenger receptors binding lipids and glycoproteins respectively, with important functions in the clearance of apoptotic cells and inflammatory debris (14, 26, 27). These were both steadily up-regulated over time by WT but not CGD MoMacs ( Figure 1B ; Supplementary Figure 2 ). Increases in size and granularity measured by forward and side-scatter (FSC, SSC) are hallmarks of macrophage differentiation from monocytes. In this model, there was a less consistent association between FSC and SSC characteristics and MoMac maturation ex vivo, possibly because MoMac morphology can be altered by adhesion to plastic tissue culture plates ( Supplementary Figure 2 ). Additional phenotyping markers were also explored ( Supplementary Figure 3 ): staining for CD226, CCR2, CD11c and Tim4 was lacking on WT and CGD MoMacs, and expression of CD11b, CD115, CD14, CX₃CR1, MHCII and MerTK did little to distinguish between genotypes ex vivo and were not explored in further analyses.

To aid the visualization of the phenotypic differences described by the marker panel in Figure 1B , we used tSNE analysis of pooled MoMac samples after normalizing for differences in cell numbers between timepoints and genotypes. This analysis confirmed the similarity of WT and CGD MoMacs at the time of plating, but showed a strong separation of the two genotypes after the first 24h in culture that persisted over at least 3 days in culture ( Figure 1C ). This separation was initially the result of the up-regulation of CD54 and CD80 by CGD MoMacs and was later strengthened by the down-regulation of Ly6C and up-regulated expression of the pro-resolving markers CD64, CD36 and CD206 by WT cells.

To validate the use of this panel in vivo we collected samples of peritoneal lavage cells from WT and CGD mice at 24 or 72 hours following intraperitoneal zymosan injection ( Figure 2A ). As expected, WT MoMacs down-regulated expression of Ly6C and up-regulated expression of F4/80, CD64, CD36 and CD206 while CGD MoMacs do not ( Figure 2B ; Supplementary Figure 4 ). Of note, expression of CD54 and CD80 was not detected as strongly in vivo, perhaps because this population mostly consists of monocytes only recently recruited from circulation as previously proven by PKH26 labeling and tracking (17). tSNE analysis again showed that 24h MoMacs from WT and CGD mice and 72h CGD MoMacs cluster relatively close together in tSNE space due to their shared expression of an “immature” marker profile whereas 72h WT MoMacs separated into a unique cluster ( Figure 2C ). This divergence represented the loss of Ly6C by WT MoMacs and the concurrent gain of pro-resolving markers particularly CD36 and CD206 whereas the 72h CGD MoMacs continued to resemble the early, pro-inflammatory MoMacs as previously described.

Altogether, these data show that ex vivo MoMac maturation, and failure to mature in CGD, leads to similar phenotypic changes to those observed in vivo as described by the expression of well-defined monocyte/macrophage proteins. These data demonstrate that the culture of total peritoneal cells is sufficient to substantially recreate the different environments that influence MoMac maturation in the peritoneal cavity and to allow identification of key factors preventing MoMac maturation in CGD.

We hypothesized that the critical cues are secreted, soluble factors produced by lavage cells and hence should accumulate in culture media over time. To this end, conditioned media was collected from plated WT or CGD lavage cells after 24h in culture and this was used to treat a second set of plated lavage cells of either genotype. Treatment of WT MoMacs with CGD-conditioned media prevented the up-regulation of F4/80, CD64, CD36 and CD206, and induced up-regulation of CD54 ( Figures 3A, B ; Supplementary Figure 5 ), indicating that normal maturation was inhibited and MoMacs instead showed pro-inflammatory activation. Treatment of CGD MoMacs with WT-conditioned media had no effect on their phenotype, suggesting that there are no soluble pro-maturing signals produced by WT cells that are able to overcome the pro-inflammatory programming of CGD cells. This was quantified using tSNE analysis of pooled MoMacs. The mature population was defined using the untreated WT MoMac population and a gate was drawn to quantify the percentage of cells from each sample that clustered together with the mature cells ( Figures 3B, C ). This analysis clearly demonstrates that treatment of WT MoMacs with CGD conditioned media completely prevented their normal maturation, while WT conditioned media had no effect on CGD MoMac phenotype. This suggested the production of inhibitory factors by CGD cells in culture that act upon both CGD and WT MoMacs. Notably, treatment with proteinase and boiling eliminated the inhibitory effect of CGD conditioned media, suggesting that one or more protein factors were responsible for inhibition of maturation ( Figure 3C ).

CGD cells are known to overproduce a number of pro-inflammatory cytokines in response to PAMP stimulation that contribute substantially to the heightened inflammatory milieu in CGD mice. Of these, TNFα is considered a master regulator of pro-inflammatory cytokine production driving NF-κB and AP-1 activation and downstream pro-inflammatory gene transcription (28), and myeloid cells from CGD patients and animals show both heightened and prolonged production of TNFα in response to fungal particles (13, 29, 30). Elevated concentrations of TNFα were detected in conditioned media from CGD cultures after 48h compared to WT (<130 vs 17,573 ± 2,516 pg/mL). To determine its role in our ex vivo model, and to establish the cellular source of TNFα, intracellular cytokine staining was performed after 24h in culture. It was found that both CGD neutrophils and macrophages continue to produce TNFα over time with detectable amounts produced spontaneously between 20 and 24h in the presence of Brefeldin A. Additionally, a greater proportion of CGD cells remained responsive to re-stimulation ( Figures 4A, B ), producing abundant TNFα in response to brief LPS stimulation.

Treatment of WT cells with increasing concentrations of recombinant TNFα significantly inhibited the maturation of WT MoMacs, most noticeably maintaining Ly6C expression, inducing CD54 expression and preventing the expression of CD36 and CD206 ( Figures 4C, D ; Supplementary Figure 6A ). Conversely, neutralization of TNFα with blocking antibody was sufficient to restore expression of F4/80, CD64, CD36 and CD206 by CGD MoMacs, so that they more closely resembled WT MoMacs than CGD MoMacs treated with control antibody ( Figures 4E, F ; Supplementary Figure 6B ). Neither treatment with exogenous TNF or neutralizing antibody significantly affected the survival of cultured leukocytes ( Supplementary Figure 7 ).

TNFα neutralization significantly decreased the production of other pro-inflammatory cytokines IL-6 and IL-1β and possibly TNFα itself ( Figure 4G ) and the neutrophil chemoattractant CXCL1 ( Supplementary Figure 8 ) suggesting that these are regulated down-stream of TNFα signaling. Concentrations of IFNγ, GM-CSF and IL-10 were also found to be elevated in CGD cultures relative to WT but were unaffected by TNFα neutralization ( Supplementary Figure 8 ). Expression of these seems to be regulated independently of TNFα and were not important determinants of MoMac maturation in this model system. Antibody-mediated blockade of IL-1β, IL-6, IFNγ, or type I interferon signaling alone had no effect on CGD MoMac maturation ( Supplementary Figure 9 ). Combination blockade of TNFα and IL-1β simultaneously had no additional effect beyond that seen with TNFα neutralization alone (data not shown). These data collectively suggest that TNFα is the predominant driver of pro-inflammatory MoMac programming in the CGD environment promoting both the maintenance of immature phenotype and pro-inflammatory cytokine production.

To assess the role of TNFα in vivo we delivered neutralizing antibody to CGD mice at 20 and 44 hours after i.p. zymosan treatment and collected peritoneal lavage to assess MoMac phenotype at 72h post-zymosan ( Figure 5A ). Cell numbers or composition in lavage were not significantly altered by anti-TNFα treatment ( Figure 5B ) but the population of lavagable MoMacs differed between anti-TNFα or isotype treated CGD mice. Greater heterogeneity exists in the WT MoMac population in vivo as monocytes continue to be recruited into the peritoneal cavity, albeit in low numbers, and so mature cells were defined in tSNE analysis using the Ly6C^lo CD64^hi CD206⁺ CD36⁺ population from WT lavage for comparison to CGD MoMacs. Approximately half of MoMacs from TNFα-neutralized CGD mice adopted a phenotype that more closely resembled WT MoMacs ( Figures 5C, D ) showing downregulation of CD54 and Ly6C, along with upregulation of CD36 and CD206, although not to quite the extent of WT MoMacs, and upregulation of CD64 above that seen in the WT MoMacs ( Supplementary Figure 10 ).

This observation supports our ex vivo data demonstrating a key role of TNFα in the impaired maturation of MoMacs in CGD mice, although it also suggests that additional factors likely exist in the more complex environment of the peritoneal cavity.

Since TNFα neutralization was able to promote phenotypic maturation we next tested whether it was able to restore efferocytosis, an important pro-resolving function of mature macrophages. Efferocytosis has been shown to be deficient in CGD macrophages (15, 16, 31, 32) and to be inhibited in other macrophage systems by TNFα (33–35). As shown in Figure 6 , 20h WT MoMacs allowed to mature for 2 days ex vivo or cultured with additional exogenous TNFα to inhibit their maturation were tested for their ability to efferocytose apoptotic neutrophils. TNFα significantly reduced the ability of these cells take up apoptotic neutrophils. This appeared to be due to TNFα-induced programming rather than acute effects on efferocytosis that have been described with short-term stimulation (36, 37) as stimulation with TNFα concurrent with apoptotic cell introduction had no effect on uptake (not shown). Conversely, 20h CGD MoMacs cultured for the same time remained immature in phenotype and significantly poorer in their capacity to take up apoptotic neutrophils. Note that the data shown were normalized to the average engulfment of untreated WT MoMacs within each experiment to account for heterogeneity in the preparation of apoptotic neutrophils. As hypothesized, neutralization of TNFα with antibody enhanced both their maturation and efferocytic capacity. Hence, the neutralization of TNFα permitted both the phenotypic maturation of CGD MoMacs and improvements in efferocytic function, a key behavior of mature pro-resolving macrophages.

WT and CGD MoMacs express both receptors for TNFα: TNFR1 and TNFR2 ( Supplementary Figure 11 ). While surface staining for both receptors was significantly lower on CGD MoMacs at the time of plating, culture induced up-regulation of both receptors in both genotypes such that receptor expression was similar after 48h ex vivo. Soluble TNFα activates TNFR1 signaling more efficiently than TNFR2 so we hypothesized that TNFR1-deficient MoMacs would be less susceptible to the pro-inflammatory environment created by CGD PL cells. In the presence of conditioned media from CGD PL cells, TNFR1^-/- MoMac maturation was largely unaffected while WT MoMac maturation was inhibited as previously observed ( Figures 7A, B ) Confirming the role of TNFα in this setting, neutralization of TNFα during conditioned media generation was sufficient to mostly restore the maturation of WT MoMacs such that they largely resembled the treated TNFR1^-/- MoMacs ( Supplementary Figure 12 ).

In direct co-culture experiments WT or TNFR1^-/- cells were plated with increasing proportions of CGD lavage cells (70:30 or 30:70 WT or TNFR1^-/-: CGD) maintaining a constant cell density. PBSE labeling was used to distinguish cells of different genotypes such that the effect of co-culture specifically on the WT or TNFR1^-/- MoMacs could be determined. Replicate experiments using different labeling protocols confirmed that PBSE had no effect on the observed phenotype or tSNE clustering. TNFR1^-/- MoMacs clustered with untreated mature WT cells while the maturation of TNFR1-sufficient WT MoMacs was inhibited ( Figures 7C, D ; Supplementary Figure 12 ) Together these data confirm that soluble TNFα, detected via TNFR1 signaling, maintains the pro-inflammatory phenotype of MoMacs in the presence of CGD myeloid cells and prevents the acquisition of a pro-resolving macrophage phenotype. Importantly, these observations are independent of any potential effects of antibody-Fc receptor binding by MoMacs previously suggested (38, 39).

To further demonstrate the necessity of TNFα:TNFR1 signaling in vivo, adoptive transfer experiments were performed where WT or TNFR1^-/- peritoneal lavage cells collected at 18h post-zymosan were transplanted into the peritonea of inflamed CGD recipient mice ( Figure 8A ). The maturation of transferred MoMacs was assessed at 72h post-zymosan (54h after adoptive transfer). As in the ex vivo experiments, adoptive transfer of WT MoMacs into CGD recipients prevented their normal maturation, induced expression of CD54, prevented down-regulation of Ly6C, and up-regulation of CD64, CD36, and CD206. ( Figures 8B, C ; Supplementary Figure 13 ). Adoptively transferred TNFR1^-/- MoMacs were not prevented from maturing and underwent down-regulation of Ly6C and up-regulation of CD64, CD36, CD206 and MHCII ( Supplementary Figure 13 ). Using tSNE analysis, the phenotype of TNFR1^-/- MoMacs showed significant overlap with that of mature MoMacs from control WT mice treated with zymosan alone. These observations support the conclusion of our ex vivo experiments that TNFα: TNFR1 signaling is critical in maintaining the pro-inflammatory phenotype of MoMacs in CGD, and that blockade of this signaling permits MoMac maturation.