HOST RESPONSE AND INFLAMMATION

crossm Distinct Effects of Integrins ␣X␤2 and ␣M␤2 on Leukocyte Subpopulations during Inflammation and Antimicrobial Responses Samir Jawhara,* Elzbieta Pluskota, Wei Cao,* Edward F. Plow, Dmitry A. Soloviev Department of Molecular Cardiology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA

ABSTRACT Integrins ␣M␤2 and ␣X␤2 are homologous adhesive receptors that are expressed on many of the same leukocyte populations and bind many of the same ligands. Although ␣M␤2 was extensively characterized and implicated in leukocyte inflammatory and immune functions, the roles of ␣X␤2 remain largely obscure. Here, we tested the ability of mice deficient in integrin ␣M␤2 or ␣X␤2 to deal with opportunistic infections and the capacity of cells derived from these animals to execute inflammatory functions. The absence of ␣M␤2 affected the recruitment of polymorphonuclear neutrophils (PMN) to bacterial and fungal pathogens as well as to model inflammatory stimuli, and ␣M␤2-deficient PMN displayed defective inflammatory functions. In contrast, deficiency of ␣X␤2 abrogated intraperitoneal recruitment and adhesive functions of monocytes and macrophages (M␾) and the ability of these cells to kill/phagocytose Candida albicans or Escherichia coli cells both ex vivo and in vivo. During systemic candidiasis, the absence of ␣X␤2 resulted in the loss of antifungal activity by tissue M␾ and inhibited the production of tumor necrosis factor alpha (TNF-␣)/interleukin-6 (IL-6) in infected kidneys. Deficiency of ␣M␤2 suppressed M␾ egress from the peritoneal cavity, decreased the production of anti-inflammatory IL10, and stimulated the secretion of IL-6. The absence of ␣X␤2, but not of ␣M␤2, increased survival against a septic challenge with lipopolysaccharide (LPS) by 2-fold. Together, these results suggest that ␣M␤2 plays a primary role in PMN inflammatory functions and regulates the anti-inflammatory functions of M␾, whereas ␣X␤2 is central in the regulation of inflammatory functions of recruited and tissue-resident M␾.

Received 26 July 2016 Returned for modification 12 August 2016 Accepted 20 October 2016 Accepted manuscript posted online 31 October 2016 Citation Jawhara S, Pluskota E, Cao W, Plow EF, Soloviev DA. 2017. Distinct effects of integrins αXβ2 and αMβ2 on leukocyte subpopulations during inflammation and antimicrobial responses. Infect Immun 85:e00644-16. https:// doi.org/10.1128/IAI.00644-16. Editor George S. Deepe, University of Cincinnati Copyright © 2016 American Society for Microbiology. All Rights Reserved. Address correspondence to Dmitry A. Soloviev, [email protected].

* Present address: Samir Jawhara, University of Lille 2, Lille, France; Wei Cao, Case Western Reserve University, Cleveland, Ohio, USA.

KEYWORDS Candida albicans, Escherichia coli, animal models, cytokines, infection control, inflammation, integrins, macrophages, monocytes, neutrophils

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ntegrins are a large family of heterodimeric adhesive cell receptors that mediate a wide spectrum of cell-cell and cell-extracellular matrix (ECM) interactions (1). They are present on cell surfaces in a conformation in which they exhibit a relatively low affinity for their cognate ligands but become activated by environmental agonists to elicit their biological functions. The ␤2 integrin subfamily, expressed primarily by leukocytes, is composed of four members, which share a ␤2 (CD18) subunit. This subunit associates noncovalently with one of four distinct but structurally homologous ␣ subunits to form ␣M␤2 (CD11b/CD18, Mac-1, or CR3), ␣X␤2, (CD11c/CD18, p150,95, or CR4), ␣L␤2 (CD11a/ CD18 or LFA-1), and ␣D␤2 (CD11d/CD18) (for reviews, see references 2 and 3). One of the most extensively characterized ␤2 integrins is ␣M␤2, which is expressed on polymorphonuclear neutrophils (PMN), monocytes, macrophages (M␾), some subsets of cytotoxic T lymphocytes, and NK cells and has been implicated in the diverse responses of these cells, including chemotaxis, inflammation, phagocytosis, and cell-mediated killing (4–6). ␣M␤2 supports these leukocyte functions by virtue of its ability to recognize and mediate responses to more than 50 structurally unrelated ligands, including January 2017 Volume 85 Issue 1 e00644-16

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fibrinogen, complement iC3b, intracellular cell adhesion molecule 1 (ICAM-1), CD40L, blood coagulation factor X, denatured proteins, as well as numerous bacterial and fungal products (7–9). Three distinct binding regions within ␣M␤2 mediate the recognition of these diverse ligands: (i) the ␣M I domain, an ⬃200-amino-acid region close to the NH2-terminal end of the integrin ␣M subunit; (ii) the ␤2 I-like domain within the ␤2 subunit; and (iii) the lectin domain, which is located in proximity to the transmembrane segment of the ␣M chain. Both the ␣M I domain and the ␤2 I-like domain (also referred to as the A domains) contain a metal ion-dependent adhesion site (MIDAS motif) in which a bound divalent cation is involved in the binding of protein/polypeptide ligands (10). Ligation of the ␣M I domain promotes cell adhesion and phagocytosis, while binding to the I-like ␤2 domain modulates cell motility and chemotaxis (4). The lectin domain of the ␣M subunit mediates the recognition of bacterial lipopolysaccharide (LPS), fungal glycans, and mannoproteins without requiring the prior activation of ␣M␤2; regulates leukocyte activation and cytokine secretion; and functions in a manner similar to that of pattern recognition receptors (PRR) such as Toll-like receptor 2 (TLR2), TLR4, or dectin-1 (8, 11, 12). Integrin ␣X␤2 is present on many of the same leukocyte subsets as ␣M␤2 but is absent on lymphocytes and is expressed at much higher levels on dendritic cells (DC), which express no ␣M␤2. Indeed, ␣X␤2 is often used as a surface marker for DC. The ␣X and ␣M chains are ⬃70% identical. The ␣X chain of ␣X␤2 integrin contains the ␣X I domain, which is involved in ligand recognition (13). While it was previously thought that ␣X␤2 lacked a lectin binding function, we found that the ␣X subunit is also able to bind fungal glycans (14). Most ␣M␤2 ligands, notably fibrinogen, ICAM-1, and iC3b, are also recognized by ␣X␤2 (13). Integrin ␣X␤2 is involved in the macrophage-mediated elimination of several bacterial and fungal pathogens and may play a role in the development of gastric ulcers in chronic Helicobacter pylori infection (14–16). It was shown previously that mice deficient in ␣X␤2, but not in ␣M␤2, developed aggravated Lyme carditis upon infection with the spirochete Borrelia burgdorferi (17). As complement receptors, both integrins are involved in the complement-dependent elimination of Gram-positive (and thus lacking LPS) capsular Streptococcus pneumoniae (18), and the two integrins do not participate in the pathogenesis of parasites such as the malaria parasite Plasmodium (19). Overall, the functions of ␣X␤2 are understudied, and its role in inflammation and infection is still unclear. A major unresolved question is why these two clearly distinct yet highly homologous receptors, ␣X␤2 and ␣M␤2, with almost identical ligand repertoires are present on many of the same leukocyte subsets. This work attempts to delineate and distinguish the individual contributions of ␣M␤2 and ␣X␤2 to the immune and inflammatory functions of different subsets of leukocytes. Mice in which the genes for ␣M␤2 (Δ␣M) or ␣X␤2 (Δ␣X) had been inactivated have been used in different models of fungal and bacterial infections. Candida albicans and Escherichia coli were selected as opportunistic fungal and bacterial pathogens, respectively. The ability of the ␤2 integrins to directly recognize these opportunistic pathogens, C. albicans via ␤-glucans or Pra1 mannoprotein (14, 20, 21) and E. coli via LPS (22–24), was an additional consideration in choosing these models of infection. RESULTS Ex vivo studies of leukocyte functions. In the experiments described in this section, we sought to compare the contribution of integrins ␣M␤2 and ␣X␤2 to the ex vivo functions of populations of immune cells obtained as thioglycolate-elicited PMN, monocytes, monocyte-derived inflammatory M␾ (iM␾), residential intraperitoneal M␾ (rM␾), as well as residential M␾ obtained from other tissues. Utilization of ␣X␤2 and ␣M␤2 for leukocyte recruitment and adhesion. Activated PMN were recovered by lavage from the inflamed peritoneal cavity 6 to 8 h after thioglycolate stimulation, while iM␾/monocytes were obtained at 72 h (21, 25, 26). In wild-type (WT) mice at 6 to 7 h, PMN constituted 90% to 95% of all cells in the lavage fluid, identified as Ly6G⫹⫹ cells by flow cytometry (Fig. 1A, left). The content of PMN in the lavage fluid from ␣M-deficient mice was reduced and constituted 40 to 45% of the January 2017 Volume 85 Issue 1 e00644-16

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FIG 1 Leukocyte recruitment into murine peritoneal cavities in response to inflammatory or infectious stimuli. (A to D) Flow cytometry for surface markers of murine PMN (Ly6G) (A and D) and M␾ (F4/80) (B and C) in cells recovered from intraperitoneal lavage fluids of WT (left), Δ␣M (middle), and Δ␣X (right) mice, obtained 6 h (A and B) or 72 h (C and D) after thioglycolate injection. The percentage of individual leukocyte subsets in the lavage fluid was calculated as a percentage of Ly6G⫹⫹ or F4/80⫹⫹ cells in total pooled lavage fluids (n ⫽ 5) by flow cytometry (M1 fraction). (E to G) Migration of leukocytes into peritoneal cavities of WT, Δ␣M, and Δ␣X mice in response to intraperitoneal injection of thioglycolate (E), C. albicans (106 cells) (F), or E. coli (107 cells) (G). The cells were harvested by lavage of the peritoneal cavity, pooled (n ⫽ 5), and counted in a hemacytometer. The data are expressed as means ⫾ SD from two representative independent experiments, each performed in triplicate. * indicates a P value of ⬍0.05 and ** indicates a P value of ⬍0.02 by ANOVA.

cell pool, while recruitment of PMN from ␣X-deficient mice was similar to that of WT PMN (Fig. 1A, right), consistent with a critical role of ␣M␤2 but not ␣X␤2 in PMN recruitment (27, 28). Neither deficiency affected the F/80⫹⫹ rM␾ population: the contents of these cells in lavage fluids from all 3 mouse strains at 6 to 8 h were similar and constituted 10% to 15% of the total cells in the lavage fluid (Fig. 1B). In contrast, at 72 h post-thioglycolate injection, when iM␾ monocytes and lymphocytes are the dominant cells in the peritoneal cavity (40 to 50% of the total cell population by flow cytometry), the content of Δ␣X iM␾ in the lavage fluid was reduced almost 3-fold to 15 to 20% of total cells (P ⬍ 0.02 by analysis of variance [ANOVA]) compared to both WT and Δ␣M iM␾, which showed a similar recruitment of F4/80⫹⫹ cells (Fig. 1C and E). January 2017 Volume 85 Issue 1 e00644-16

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Fewer than 10% of cells in the 72-h lavage fluid were PMN (Ly6G⫹⫹ cells) in all 3 strains (Fig. 1D). We also confirmed data from a recent report (29) showing that the elimination of ␣M␤2 did not halt the recruitment of PMN to the peritoneal cavity but rather delayed the response, which reached a maximum at 16 to 18 h, versus 6 to 8 h in WT and Δ␣X mice (data not shown). To evaluate the roles of ␣M␤2 and ␣X␤2 in leukocyte recruitment to more biologically relevant stimuli, we inoculated the three mouse strains by intraperitoneal (i.p.) injection of C. albicans or E. coli. Components of these pathogens are recognized not only by the ␤2 integrins. Leukocytes can also be activated via different PRRs: TLR4 binds bacterial LPS (30, 31), and TLR2/dectin-1 interacts with fungal glycans (32). Therefore, these pathogens represent complex as well as physiologically relevant stimuli. Nevertheless, the patterns of leukocyte recruitment to the site of C. albicans (Fig. 1F) or E. coli (Fig. 1G) infection were extremely similar to the pattern of recruitment observed with thioglycolate-induced inflammation (Fig. 1E), and lavage fluids contained similar leukocyte subsets, as confirmed by flow cytometry (results not shown). In contrast, deficiency of ␣X hampered only M␾ influx into the peritoneal cavity. The absence of either integrin did not affect lymphocyte recruitment to either pathogen (Fig. 1E to G). Next, we evaluated the adhesive properties of the leukocytes isolated from peritoneal lavage fluids of all 3 mouse strains, including rM␾ harvested from nonstimulated mice (0-h lavage fluid). Cells in the 6-h lavage fluid were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ly6G (for PMN isolation), cells from 0-h and 72-h lavage fluids were stained with FITC-labeled anti-mouse F4/80 (for M␾), and leukocytes with similar levels of these antigens were sorted by fluorescence-activated cell sorter (FACS) analysis and used immediately for all further experiments ex vivo (Fig. 2A to E). Since leukocyte binding to fibrinogen via ␣M␤2 is a requisite for successful intraperitoneal clearance of Staphylococcus aureus (33), the fibrinogen D100 fragment was used as an activation-specific protein ligand for both ␣M␤2 and ␣X␤2 integrins (34–36). The small peptide ligand P2C, comprising a ␤2 integrin recognition sequence within the fibrinogen ␥-chain (35) that can support ␤2 integrin-dependent leukocyte adhesion regardless of the activation state of the integrins (D. Soloviev, unpublished observations), was used to corroborate the function of integrins expressed on the surfaces of cells. TΔ␣M PMN failed to adhere to the immobilized D100 fragment, while the adhesion of Δ␣X PMN was similar to that of WT cells. In contrast, Δ␣X iM␾ did not adhere to this ligand, while Δ␣M and WT iM␾ adhered equally well (Fig. 2B). P2C supported the adhesion of PMN, rM␾, and iM␾ derived from all three mouse strains, verifying the capacity of the integrins, ␣X␤2 on the surface of Δ␣M PMN and ␣M␤2 on the surface of Δ␣X rM␾, to support adhesive functions (Fig. 2C). In contrast, the levels of binding of rM␾ from all 3 mouse strains to both ligands were similar and reached 40% and 80% of the adhesion of iM␾ to D100 and P2C, respectively (Fig. 2B and C). These data indicate that iM␾ utilize ␣X␤2 for adhesion, while PMN engage these ligands via ␣M␤2. PMN utilize ␣M␤2, and iM␾ require ␣X␤2 for phagocytosis and successful intracellular killing of pathogens. The participation of each of the ␤2 integrins in phagocytosis/killing was initially tested ex vivo by coincubation of isolated leukocyte subpopulations with a pathogen. In these experiments, we utilized inflammatory peritoneal leukocytes recruited in response to thioglycolate. These leukocytes had been activated for several hours (PMN) to several days (M␾) prior to their isolation and are known to be depleted of most of their granule content during their migration to the peritoneal cavity (21, 37). Therefore, after transmigration, isolation, and washing, PMN and iM␾ were significantly depleted of extracellular killing capabilities, and the most prominent residual antimicrobial activity would be mediated by the phagocytosis of opsonized or nonopsonized pathogens with subsequent intracellular killing. ␣X␤2 and ␣M␤2 are pivotal receptors for the phagocytosis of opsonized pathogens, and we have demonstrated both in vitro and in vivo that, in the absence of complement, leukocytes can successfully engulf and kill nonopsonized C. albicans cells only via a ␤2-integrindependent mechanism. This mechanism depends on the recognition of PRA1, a fungal January 2017 Volume 85 Issue 1 e00644-16

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FIG 2 Adhesive and phagocytic/intracellular killing functions of isolated leukocytes. (A) Dual-fluorescent staining of cells sorted by flow cytometry for PMN (surface markers Ly6G and CD18) (top) and M␾ (surface markers F4/80 and CD18) (bottom) from WT (left), Δ␣M (middle), and Δ␣X (right) mice. (B and C) Adhesion of rM␾, PMN, and iM␾ isolated by flow cytometry from WT, Δ␣M, and Δ␣X mice to the D fragment of human fibrinogen (B) or to the P2C peptide ligand (C). Activation of integrin ␣M␤2 or ␣X␤2 is a prerequisite for adhesion to protein ligands, represented by the fibrinogen D fragment, but recognition of P2C, a peptide sequence in fibrinogen recognized by ␣M␤2 and ␣X␤2, does not require activation and was used to validate that the integrins are present and functional. (D and E) Phagocytosis of C. albicans by PMN (D) or iM␾ (E) isolated from WT, Δ␣M, and Δ␣X mice. Phagocytosis by leukocytes, which were additionally prestimulated with PMA to activate expressed but not activated ␤2 integrins, is shown as black bars. (F) Impact of 2 ␮M ouabain, anti-␤2 MAb M18/2, and control rat Ig on phagocytosis/killing of C. albicans by PMN (left) or M␾ (right). The positive control (no inhibitors) is shown as black bars, and the negative control (no cells) is shown as gray bars. All data are presented as means ⫾ standard errors of the means for triplicate samples from at least three independent experiments. Asterisks indicate a significant difference (*, P ⬍ 0.05 by ANOVA).

mannoprotein which is expressed exclusively on the surface of fungal germ tubes and hyphae (20, 38). To commence this assay, isolated PMN or iM␾ were coincubated at a ratio of 7:1 with germinated C. albicans cells for 2 h, and residual viable fungal cells were enumerated as CFU. With WT PMN, only 25 to 30% of the fungi remained viable, and Δ␣X PMN January 2017 Volume 85 Issue 1 e00644-16

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showed a similar potency in antifungal activity. In contrast, the antifungal activity of Δ␣M PMN was significantly reduced (P ⬍ 0.01), as 75% of the fungi remained viable (Fig. 2D). When C. albicans cells were incubated with iM␾, Δ␣X cells were inept in C. albicans removal (70% ⫾ 5% survival of the fungus), while only 30% ⫾ 10% of the fungi remained viable after incubation with WT or Δ␣M iM␾, indicating similar phagocytic activities of these leukocyte populations (P ⫽ 0.37) (Fig. 2E, clear, gray, and dashed bars). Both anti-␤2-blocking M18/2 monoclonal antibody (MAb) and ouabain, a known inhibitor of phagocytosis (39–41), protected the fungus against killing by PMN or iM␾ (survival of 95 to 105% of added C. albicans cells), indicating that killing was ␤2 dependent and phagocytosis dependent. MAb M18/2 produced this inhibition at 10 ␮g/ml, while an isotype-matched control MAb had no effect (Fig. 2F). Ouabain was effective at 2 ␮M, a concentration known to block phagocytosis (39, 41). Taken together, the above-described data indicate that for phagocytosis, as for migration and adhesion, PMN utilize ␣M␤2, and iM␾ utilize ␣X␤2. However, the residual abilities of both Δ␣M PMN and Δ␣X rM␾ to adhere to the D100 fragment suggest that minor fractions of their ␤2 integrin counterparts are at least partially functional. Thus, next, we determined the ability of thioglycolate-activated leukocytes, Δ␣M PMN and Δ␣X iM␾, to additionally activate ␣X␤2 or ␣M␤2. In these experiments, the thioglycolateelicited leukocyte subsets from all 3 mouse strains were preactivated with 0.2 nM phorbol myristate acetate (PMA) and incubated with the fungus for 2 h. PMA stimulation did not affect C. albicans killing by WT (control) and Δ␣X PMN, and PMA-treated Δ␣M PMN did not significantly decrease fungal survival (P ⫽ 0.093) (Fig. 2C, black bars). In contrast, WT and Δ␣X iM␾, but not Δ␣M iM␾, showed a significant (P ⬍ 0.05) boost in phagocytic activity upon treatment with PMA, decreasing fungal viability from 70% to 40 to 45% (Fig. 2D, black bars). These data suggest that while M␾ engage ␣X␤2 as a primary mediator of fungal killing, they are still able to utilize ␣M␤2 if appropriately activated. In vivo studies using murine models of infection and inflammation. (i) Intraperitoneal iM␾ engage ␣X␤2 to eliminate C. albicans in vivo. Whereas thioglycolate-activated leukocytes rely almost exclusively on phagocytosis to eliminate pathogens ex vivo, in vivo, they deploy multiple mechanisms to achieve cytotoxicity (e.g., oxidative burst or secretion of proteases, defensins, lysozyme, peroxide, or complement [42, 43]). As previously shown, the ␤2 integrins also play a role in the regulation of leukocyte secretion, and a deficiency of ␣M results in defective PMN degranulation (21, 37). To assess the contribution of the individual ␤2 integrins to the cytotoxic function of intraperitoneal PMN and M␾ in vivo, we utilized a modified two-stage method of intraperitoneal candidiasis in a presensitized peritoneum (44). In this assay, mice were initially stimulated with thioglycolate for 6 and 72 h (WT and Δ␣X mice) or 16 h and 72 h (Δ␣M mice) to elicit the recruitment of the specific leukocyte subsets before intraperitoneal infection with the pathogen. The 16-h time point, when the content of Δ␣M PMN in the thioglycolate-stimulated peritoneum is maximal (29), was chosen instead of the 6-h time point to ensure that any changes in activity were not a consequence of low PMN levels. Two hours after injection of C. albicans, mice were euthanized; cells were recovered by peritoneal lavage, washed, and lysed with 1% Tween 80; and the amounts of surviving pathogens were enumerated as CFU upon plating of dilutions of the lavage fluids onto agar plates. Under these conditions, the cellular pool in the nonstimulated peritoneum of WT mice, consisting predominantly of rM␾ and B lymphocytes (45), together with noncellular mechanisms, decreased C. albicans clearance by one-half (52% ⫾ 11%; P ⬍ 0.005). Genetic elimination of either ␤2 integrin significantly (P ⬍ 0.05 by ANOVA) hampered the antifungal activity of rM␾ and thereby increased the survival of C. albicans to 65 to 70% in the nonstimulated peritoneal cavities of either Δ␣M or Δ␣X mice (P ⬍ 0.05), with insignificant differences between individual integrins (P ⫽ 0.187 by t test) (Fig. 3A). The PMN (6-h intraperitoneal cells) from WT and Δ␣X mice exhibited reduced and similar fungal clearance rates, 21% and 25%, respectively (P ⫽ 0.52), compared to the challenging inoculum. Unlike Δ␣M mice, the 6-h intraperitoneal pool of cells from Δ␣M mice, January 2017 Volume 85 Issue 1 e00644-16

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FIG 3 Effect of integrin ␣M␤2 or ␣X␤2 on the antipathogen activities of different leukocyte populations in two distinct murine models in vivo. (A and B) Model of peritonitis in a presensitized peritoneum. Mice of the WT, Δ␣M, and Δ␣X strains were stimulated by intraperitoneal injection of thioglycolate to initiate leukocyte recruitment. Six hours and 72 h after thioglycolate injection, the mice (n ⫽ 5) were challenged with C. albicans (A) or E. coli (B), and after 3 h, clearance of the pathogens was determined by measuring the CFU in intraperitoneal lavage fluids. (C) Classical model of fungal peritonitis. Mice (n ⫽ 5) of the WT, Δ␣M, and Δ␣X strains were challenged intraperitoneally with C. albicans. Six hours, 16 h, and 72 h after infection, intraperitoneal clearance was determined. Each bar represents the mean ⫾ SD for triplicate samples from at least two independent experiments. Significance was determined by comparing WT to deficient mice at each time point. Asterisks indicate a significant difference (*, P ⬍ 0.05 by ANOVA).

in which PMN recruitment was attenuated, was able to decrease fungal clearance only to 56% ⫾ 12%. Since this level of antifungal activity was similar to the activity of WT rM␾ (P ⫽ 0.087), the intraperitoneal residential cells appeared to be primarily responsible for the antifungal activity in the 6-h Δ␣M cell pool. This result suggests that recruited Δ␣M PMN in the 6-h intraperitoneal milieu are unable to control fungal growth. This interpretation is further supported by the finding that cells derived from Δ␣M mice 16 h after infection, which contain 4- to 5-fold more PMN, demonstrated a clearance rate of 46%, similar to that of the 6-h pool (P ⫽ 0.18). Seventy-two hours after thioglycolate stimulation, the intraperitoneal pool of cells consisted of approximately equal numbers of iM␾ and lymphocytes (Fig. 1A). The cells in the WT and Δ␣M cell pools reduced fungal clearance to 22% to 26%. In contrast, Δ␣X iM␾ failed to clear C. albicans; the clearance rate was 48% ⫾ 16% (Fig. 3A). These data indicate that iM␾ utilize ␣X␤2 for both intracellular and extracellular killing of the fungus, unlike PMN, which engage ␣M␤2 for these purposes. This pattern was recapitulated with E. coli as the pathogen, except that the difference in the antibacterial activities of WT and knockout (KO) rM␾ was insignificant (P ⫽ 0.087) (Fig. 3B). Thus, ␣M␤2 on PMN and ␣X␤2 on M␾ serve as the principal receptors for representative bacterial and fungal pathogens and for inflammatory agonists (see also references 20, 43, and 46). January 2017 Volume 85 Issue 1 e00644-16

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These data were further corroborated by using a well-accepted model of acute candidal peritonitis, in which the pathogen serves as a primary inflammatory agent (47). Mice were challenged with C. albicans yeast cells by i.p. injection, and the amount of viable fungi remaining in the peritoneal cavity was determined at 6 h or 72 h postinjection. Under these conditions, at 6 h, WT mice demonstrated fungal clearance of 45% ⫾ 12% of the initial inoculum, while Δ␣M and Δ␣X mice coped with infection much less effectively, reducing intraperitoneal fungal clearance to 25% ⫾ 9% and 30% ⫾ 11%, respectively (Fig. 3C, left). After 16 h of infection, the fungal intraperitoneal burden in control and Δ␣X mice had been effectively cleared, with only 18% to 22% of the initial inoculum remaining, whereas fungal viability in Δ␣M mice remained at 52% ⫾ 12%, establishing a crucial role of ␣M in the elimination of the pathogen by PMN (Fig. 3C, middle). At 72 h, the peritoneal cavities of control and Δ␣M mice were almost completely devoid of the fungus: only 2 to 3 CFU were found in their undiluted lavage fluids, while fungal survival in Δ␣X mice was similar to that at 16 h (18% ⫾ 8%) (Fig. 3C, right), indicating a pivotal role of ␣X␤2 at later stages of the infection, when the majority of intraperitoneal cells consist of iM␾. Taken together, the data in these in vivo experiments indicate that iM␾ utilize ␣X␤2 for proinflammatory functions such as migration to the site of inflammation/infection, adhesion to ECM proteins, and pathogen elimination by extra- and intracellular mechanisms, whereas ␣M␤2 regulates these functions in activated PMN. (ii) Tissue M␾ utilize ␣X␤2 to contain fungal invasion. To examine the contribution of the two ␤2 integrins to the functions of tissue M␾, Δ␣M, Δ␣X, and WT mice were tested in a model of systemic candidiasis (48). The mice were challenged intravenously with a sublethal inoculum of C. albicans via tail vein injection and sacrificed at 16 h or 40 h postinoculation. The organs were then removed and homogenized, and organ fungal burdens (FB) were enumerated as CFU. FB at 16 h reflect fungal invasion, and FB at 40 h reflect further colonization of the organ. At 16 h, with the exception of the heart, FB in Δ␣X mice were elevated 2-fold in lungs, 3-fold in liver, and 10-fold in brain compared to the FB in WT mice. The deficiency of ␣M did not impact FB in most organs except the liver, where it was significantly increased compared to that in WT mice but was still 50% lower than that in Δ␣X mice (P ⬍ 0.05) (Fig. 4A). The low FB in lung and other vasculature-rich organs is consistent with data from a previous report showing that a deficiency of ␣M␤2 increases infiltration to the lungs after S. pneumoniae infection (49). These data indicate that integrin ␣X␤2 plays a significant role in controlling the initial stages of fungal infection (48, 50). In contrast, ␣M␤2 deficiency decreased the resistance of all organs to fungal colonization at later stages of infection. Forty hours after infection, the FB in kidneys of WT mice decreased by 8-fold compared to the FB at 16 h, and the fungus was almost completely cleared from all other organs. Δ␣X mice were able to eliminate the fungus from lungs, heart, and spleen, and the FB was decreased in kidneys to a level similar to that in WT mice, but the FB in the brain of Δ␣X mice increased by 50% (P ⬍ 0.05), and the FB in the liver remained at the same level as that at 16 h (P ⫽ 0.31). Δ␣M mice demonstrated a 1.5- to 15-fold increase in FB in all organs except spleen (Fig. 4B), indicating that ␣M␤2 is important for containing pathogen propagation in later stages of infection. The conclusion that ␣X␤2 but not ␣M␤2 is pivotal for protection by tissue M␾ was supported by data for leukocyte infiltration into infected kidneys and liver; the contents of M␾ at 16 h in liver and kidneys of all mouse strains were similar (Table 1), yet the antifungal activity of Δ␣M M␾ and Kupffer cells was significantly suppressed. On the other hand, deletion of ␣M significantly impeded PMN recruitment to infected organs, especially kidneys, which correlated with increased FB in this tissue. This depletion in functional PMN became evident even as early as 16 h and was maintained at 40 h (Table 1). (iii) Elimination of ␣X␤2 inhibits the production of the proinflammatory cytokines TNF-␣ and IL-6, and elimination of ␣M␤2 suppresses the secretion of the anti-inflammatory cytokine IL-10. As cytokines are crucial regulators of immune responses and are a hallmark of leukocyte phenotypic polarization, we examined the January 2017 Volume 85 Issue 1 e00644-16

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FIG 4 Role of integrin ␣M␤2 or ␣X␤2 in the acute phase (16 h) and the late phase (40 h) of the antifungal response to C. albicans infection. (A and B) Fungal burden in organs of WT, Δ␣M, and Δ␣X mice 16 h (A) and 40 h (B) after intravenous challenge with 105 C. albicans cells/mouse. (C and D) Levels of TNF-␣ (top), IL-6 (middle), and IL-10 (bottom) in kidney extracts derived 24 h (clear bars) (C) or 14 days (black bars) (D) after challenge with 105 C. albicans cells/mouse. The data are shown as means ⫾ SD, and each bar represents results from at least 2 independent experiments (n ⫽ 5). Asterisks indicate significant differences compared to the WT (*, P ⬍ 0.05; **, P ⬍ 0.02 [by ANOVA {A and B} or Student’s t test {C and D}]).

effect of ␣M␤2 and ␣X␤2 deficiencies on the secretion of major pro- and antiinflammatory cytokines in kidneys (51). Mice were challenged intravenously with sublethal inocula (103 cells/g) of C. albicans. The levels of tumor necrosis factor alpha (TNF-␣), interleukin-6 (IL-6), and IL-10 were determined in the kidneys (the primary target for C. albicans invasion) 24 h and 14 days after injection (48). TNF-␣ is a multifunctional proinflammatory cytokine, and activated M␾ are the main source of TNF-␣ in the kidney in response to infection (51). Elimination of ␣X␤2 significantly suppressed the production of this cytokine. Upon the onset of infection at 24 h, the level of TNF-␣ in kidneys of Δ␣X mice was 890 ⫾ 60 pg/g, which was ⬃15% lower (P ⬍ 0.05) than the level in kidneys of WT or Δ␣M mice (1,080 ⫾ 80 pg/g or 1,120 ⫾80 pg/g, respectively). On day 14, when kidneys were almost completely cleared of the fungus (0 to 50 CFU/g), the suppression of TNF-␣ production in Δ␣X mice became more apparent, and the level of TNF-␣ was 2- to 3-fold lower than that in WT or Δ␣M mice (P ⬍ 0.02). In contrast, elimination of ␣M␤2 had the opposite effect: TNF-␣ levels were increased by 20% at the 14-day time point postinfection (Fig. 4C and D). IL-6 is a cytokine which elicits both pro- and anti-inflammatory effects, but in the kidney, it promotes local inflammation by the stimulation of leukocyte recruitment, monocyte differentiation to M␾, and induction of acute-phase protein responses (51, 52). This cytokine is produced by numerous immune cells in response to several stimuli, includJanuary 2017 Volume 85 Issue 1 e00644-16

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TABLE 1 Infiltration of PMN and M␾ into murine kidney and liver 16 and 40 h after challenge with 105 C. albicans cells/mousea

Organ Kidney

Time after infection (h) 0 16 40

Liver

0 16 40

Mean no. of cells/HPF ⴞ SE

Marker (cell type) Ly6G (PMN) F8/40 (M␾) Ly6G (PMN) F8/40 (M␾) Ly6G (PMN) F8/40 (M␾)

WT mice 3⫾1 12 ⫾ 5 38 ⫾ 7 14 ⫾ 4 132 ⫾ 22 18 ⫾ 3

⌬␣M mice 2⫾1 14 ⫾ 4 18 ⫾ 6* 17 ⫾ 5 48 ⫾ 8* 21 ⫾ 7

⌬␣X mice 3⫾2 12 ⫾ 5 32 ⫾ 5 12 ⫾ 4 118 ⫾ 12 13 ⫾ 5*

Ly6G (PMN) F8/40 (M␾) Ly6G (PMN) F8/40 (M␾) Ly6G (PMN) F8/40 (M␾)

1⫾1 7⫾2 8⫾4 5⫾2 46 ⫾ 8 12 ⫾ 6

1⫾1 8⫾3 3 ⫾ 2* 9 ⫾ 3* 22 ⫾ 5* 14 ⫾ 4

1⫾1 7⫾3 7⫾4 4⫾2 42 ⫾ 8 5 ⫾ 5*

aThe

organ tissues were harvested at the indicated time points and immunostained with MAb for Ly6G (PMN) or F4/80 (M␾). The numbers of positive cells in at least eight random fields (HPF) per slide (n ⫽ 3) were counted. Data are expressed as means of 3 ⫻ 8 HPF ⫾ SE. Asterisks indicate significance (P ⬍ 0.05) between the same time/organ groups by ANOVA.

ing TNF-␣, IL-1␤, oxidative stress, or fungal glycans (53, 54). As with TNF-␣, ␣M␤2 deficiency enhanced IL-6 levels, and ␣X␤2 deficiency reduced the levels of this cytokine by 50% compared to those in WT mice at day 14 (Fig. 4D). In contrast, depletion of these integrins had an opposite effect on the anti-inflammatory cytokine IL-10. The production of IL-10 in Δ␣M mice at 24 h was decreased by 50% (1,620 ⫾ 130 pg/ml in Δ␣M mice versus 2,540 ⫾ 120 pg/ml in WT mice; P ⬍ 0.05), while IL-10 levels were similar in WT and Δ␣X mice. On day 14, Δ␣X kidneys contained 2.5-fold less IL-10 than in WT mice, while the absence of ␣M␤2 completely suppressed IL-10 levels (Fig. 4C and D). These results suggest that ␣X␤2 expression by kidney M␾ is associated with the secretion of the proinflammatory Th1 cytokines TNF-␣ and IL-6, while the expression of ␣M␤2 is associated with the secretion of the anti-inflammatory Th2 cytokine IL-10 (55). (iv) ␣X␤2 deficiency protects mice against endotoxin-induced septic shock. The proposed proinflammatory role of M␾ ␣X␤2 was further tested in a murine model of septicemia induced by i.p. injection of E. coli LPS (5 mg/kg of body weight). In this model, purified endotoxin induces acute inflammation primarily at the site of administration by rapid, local activation of immune cells, predominantly PMN, DC, rM␾, and monocytes, which initiate secondary inflammation by the secretion of a wide range of cytokines. This model is distinct from inflammatory foci caused by microbial infection of multiple organs, as is observed in the case of E. coli septicemia. As shown in Fig. 5A, upon challenge with LPS, some mice of all 3 strains died within the first 24 to 32 h postinjection, and after 3 days (72 h), 70% of both WT and Δ␣M mice succumbed. In contrast, 70% of Δ␣X mice remained viable after 72 h, and on the final, 6th day of the experiment, 60% were still alive and displayed only minor signs of distress (e.g., hunched posture and decreased mobility, etc.), scoring 0 to 5 points on our “12-point scale of distress” (21). A possible explanation for the increased survival of Δ␣X mice may be the reduced inflammatory responses of Δ␣X M␾. Indeed, images of lung sections stained with anti-F4/80 MAb showed decreased iM␾ content in Δ␣X mice compared to that in WT or Δ␣M mice (Fig. 5B and C). DISCUSSION Although several studies have compared the involvements of the ␤2 integrins in specific responses, e.g., complement recognition (18), development of Lyme carditis (17), and recognition of the malaria parasite Plasmodium (19), to our knowledge, our studies are the first attempts at an in-depth analysis of the contributions of the individual integrins to the antimicrobial and inflammatory functions of different leukocyte subsets. January 2017 Volume 85 Issue 1 e00644-16

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FIG 5 ␣X␤2 deficiency prolongs mouse survival during endotoxemia. (A) Survival of WT, Δ␣M, and Δ␣X mice upon intraperitoneal injection of LPS (5 mg/kg). P values were calculated by a Kaplan-Meier log rank test. (B) Macrophage infiltration into lungs of WT, Δ␣M, and Δ␣X mice 48 h after LPS injection. Lung sections were stained with anti-monocyte/macrophage F4/80 MAb. Arrows point to macrophages (dark brown). Bars, 50 ␮m. The photomicrographs show a representative high-power field (HPF) used for the quantitation of infiltrated M␾ in panel C. (C) Quantitation of M␾ in lungs by direct counting of F4/80-positive cells under a microscope in a number of randomly selected high-power fields. Data present the means for 5 HPF ⫾ SE. * indicates a P value of ⬍0.05 by Student’s t test.

Our data suggest that monocytes, monocyte-derived iM␾, and tissue M␾ activate and utilize ␣X␤2 for their inflammatory functions (migration, adhesion, and phagocytosis), while ␣M␤2 is required to support these same responses in PMN. The involvement of these integrins by these leukocyte subsets was consistent regardless of the January 2017 Volume 85 Issue 1 e00644-16

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inciting agent, whether the cells were activated indirectly by inflammatory cytokines or directly by bacterial or fungal ligands (56). Traditionally, integrin ␣X␤2 is considered to be a slow-activating receptor involved in delayed-type immune responses (57–59). Thus, our finding that tissue M␾ first activate and engage ␣X␤2, and not ␣M␤2, in their primary inflammatory functions is quite unexpected. While tissue M␾ are derived from local in situ proliferation of tissue monocytes or tissue-resident macrophage CFU (60), intraperitoneal inflammatory iM␾ are differentiated from circulating blood monocytes during and after their transmigration to the peritoneal cavity (61). The reduced recruitment of iM␾ by the elimination of ␣X␤2, but not of ␣M␤2, suggests that monocytes may also utilize ␣X␤2 rather than ␣M␤2 for recruitment to sites of inflammation or infection. This interpretation is consistent with previous reports demonstrating that the in vitro differentiation of PMA-activated monocytes to M␾ is associated with the induction of the ␣X promoter and upregulation of ␣X␤2 expression on the cell surface (62). The use of integrin-deficient mice in the model of systemic funginemia provided valuable information on the in vivo activities of tissue-resident M␾, which are difficult not only to examine but also to isolate. The increased fungal burden in murine organs recovered from Δ␣X mice at the onset of infection (16 h) indicated that tissue M␾ such as microglia, alveolar M␾, Kupffer cells, and kidney monocytes/M␾ engage ␣X␤2 to contain fungal invasion. In contrast, organs recovered 40 h after infection revealed a central role of ␣M␤2 in later stages of leukocyte antifungal activity. Individual phagocytic cells are able to engulf only a limited number of microbes, and since residential M␾ are present in tissues in only limited numbers and their differentiation from monocytes in situ is slow, they are unable to contain infection for a prolonged period. Migration of PMN to infected tissues, including lungs, liver, kidneys, and the central nervous system (CNS), is well documented in the literature (see, e.g., reference 63), and PMN recruitment is ultimately required to control infection. Thus, the synchronization of ␣M␤2 and ␣X␤2 functions on different leukocyte subsets is crucial for successful antimicrobial host defense. Monocytes and M␾ utilize ␣X␤2 to engulf pathogens because only integrindependent phagocytosis leads to the formation of phagolysosomes (64). This activity leads to the depletion of the ␣X␤2 cellular pool. Thus, the activation of the alternative integrin (␣M␤2) with a similar specificity may be necessary for phagocytosis as well as for subsequent M␾ efflux to present processed antigens. This conclusion of only a supplemental role of ␣M␤2 on monocyte-derived cells is supported by recent findings that indicate that TLR-dependent activation of murine DC (and, thus, by pathogen ligands) is associated with downregulation of the expression of CD11c on their surface (30, 65), enhancement of endocytosis, and modulation of antigen presentation (66). Leukocyte adhesion deficiency type 1 (LAD-1) is a hereditary disease notably characterized both by increased susceptibility to infections due to the inability to recruit leukocytes to sites of infection and by the development of autoimmune skin diseases resulting from the defective migration of tissue rM␾ and iM␾ away from sites of inflammation. The most common cases of LAD-1 arise from an absence of all 4 ␤2 integrins on leukocytes, caused by mutations in the ␤2 gene (67, 68). Particularly, low levels of CD18 (␤2) expression on leukocytes have been implicated in the pathogenesis of psoriasis and different granulomas (69). A patient with a rare variant of LAD-1 with defective CD11b expression, but normal CD18 expression, was described recently (70). This deficiency manifests in recurrent skin infections without pus formation, persistent gingivitis, periodontitis, and psoriasis. This patient showed persistent neutrophilia, reduced CD18 expression on CD4⫹ T cells (which normally express ␣L␤2 and ␣M␤2 but not ␣X␤2), and normal expression of CD18 and CD11c on PMN (70). These observations suggest the importance of ␣M␤2 but not of ␣X␤2 in the resolution of inflammation and for the inhibition of autoimmune diseases such as psoriasis associated with defective M␾ egress. The enhancement of WT and Δ␣X iM␾ emigration upon stimulation with PMA is also consistent with the necessity of ␣M␤2 for the efflux of iM␾ from the peritoneal cavity to lymph nodes as reported previously (29). The existing literature often considers thioglycolate-elicited iM␾ as being nonactiJanuary 2017 Volume 85 Issue 1 e00644-16

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vated. This supposition is based on the limited ability of these cells to kill/phagocytose bacteria compared to PMA-activated M␾ as well as on other similar observations (61, 71). Also, activation with PMA expedites the efflux of M␾ from the peritoneal cavity to lymph nodes, and the egress of PMA-activated M␾ is ␣M␤2 dependent (29). This observation is consistent with our data showing that stimulation with PMA/TNF-␣ activates ␣M␤2 on thioglycolate-induced M␾ and enhances the phagocytosis of C. albicans by WT and Δ␣X M␾ but not by Δ␣M M␾. On the other hand, the claim that thioglycolate-induced M␾ are nonactivated seems controversial, as leukocytes utilize activated ␤2 integrins for ICAM-1-dependent transendothelial migration to the peritoneal cavity, and the activation of the ␤2 integrins occurs in concert with leukocyte activation (see, e.g., reference 72). In our experiments, the migratory, adhesive, phagocytic, and antimicrobial activities of thioglycolate-induced M␾ did not differ significantly from the corresponding activities of iM␾, which were elicited and activated by bacterial or fungal ligands. Taken together, our results demonstrate that ␣X␤2 is a principal integrin receptor utilized by monocytes and M␾ and is crucial for their inflammatory and antimicrobial responses. On the other hand, PMN utilize ␣M␤2 to execute these responses. Our results also show that secretion of inflammatory cytokines such as TNF-␣ and IL-6 by renal monocytes/M␾ during the acute phase of a fungal infection and protection against death from endotoxin shock are ␣X␤2 dependent. ␣M␤2-dependent secretion of the anti-inflammatory cytokine IL-10 is prominent in later stages of resolving infections. MATERIALS AND METHODS Animals. Δ␣M and Δ␣X mice were generated in parallel with other ␤2-deficient mice and were a gift from Christy M. Ballantyne of the Baylor College of Medicine, Houston, TX (37). In numerous previous reports, it was demonstrated that the deletion of ␣M does not affect the expression of ␣X or ␣L and that the elimination of ␣X also does not affect the expression of ␣M or ␣L subunits (27, 37). However, we confirmed that the expression of ␤2 on mouse leukocytes is reduced by 30 to 40% by the deletion of either ␣ subunit (37). In our laboratory, these mice were backcrossed for more than 12 generations into a C57BL/J6 background and maintained as homozygous knockout animals. Age-matched C57BL/6J mice (Jackson Laboratories) were used as controls (WT). All protocols involving mice were approved by the Institutional Animal Care and Use Committee in accordance with Public Health Service policy, the Health Research Extension Act (Public Law 99-158), and Cleveland Clinic policy. All experiments on mice involving C. albicans or E. coli infections were carried out in a biosafety level 2 (BSL2) facility. The mice were supplied with food (Teklad diet, catalog number 2918; Harlan), had unlimited access to sterilized water, and were maintained during experiments on a 12-h alternating light/dark cycle. The mice were 10 to 12 weeks of age, were of both genders, and weighed 18 to 22 g. Candida albicans and E. coli strains. C. albicans SC5314 and E. coli K-12 (ATCC 10798) were used. Routinely, the fungus was grown on Difco Sabouraud dextrose agar (SDA; Becton Dickinson) plates as yeast cells, and E. coli was maintained on LB agar plates (Becton Dickinson). Thioglycolate-induced leukocyte recruitment to the peritoneal cavity. Leukocyte recruitment was initiated by i.p. injection of 0.5 ml of 4% thioglycolate in phosphate-buffered saline (PBS), 107 C. albicans blastoconidia, or 108 E. coli cells. Six hours or 72 h after injection, mice were euthanized by CO2 inhalation, their peritoneal cavities were opened, and intraperitoneal cells were harvested by lavage with 4 ml sterile ice-cold PBS, as described previously (73). Total cells in all lavage fluids were counted by using a hemacytometer. The content of individual leukocyte subsets was assayed by flow cytometry using a BD FACSCalibur instrument (Becton Dickinson) and FITC-conjugated rat-anti mouse Ly6G (anti-PMN) and F4/80 (anti-monocyte/M␾), all from Serotec. The lavage fluids from all 3 mouse strains, Δ␣M␤2, Δ␣X␤2, and WT, were used as a source for the isolation of individual leukocyte subsets. To isolate PMN, intraperitoneal cells were lavaged from the intraperitoneal cavity 6 h after thioglycolate administration and enriched with PMN by adhesion to plastic for 20 min at room temperature (RT). Subsequently, adherent cells were harvested by treatment with cell dissociation buffer (Thermo Fisher), washed, and labeled with anti-Ly6G-FITC MAbs. Cell pools from the 72-h lavage fluids or lavage fluids obtained from nonstimulated mice were enriched for M␾ by adhesion to plastic for 15 min and labeled with anti-F4/ 80-FITC MAbs for the isolation of inflammatory thioglycolate-elicited M␾ (iM␾) or intraperitoneal residential M␾ (rM␾), respectively. Subsequently, 5 ⫻ 106 to 2 ⫻ 107 cells were sorted by flow cytometry using a FACSCalibur cell sorter (Becton Dickinson) to obtain leukocytes of either type with similar levels of CD18 and Ly6G or F4/80 expression and immediately used in all experiments ex vivo. In control experiments, we found that the anti-Ly6G and anti-F4/80 antibodies used for PMN and M␾ sorting did not affect the adhesion of cells from unsorted WT 6-h or 72-h lavage fluids to the fibrinogen D fragment (data not shown). In some cases, thioglycolate-treated mice were additionally stimulated by i.p. injection of 0.1 ␮g PMA 4 h prior to lavage. The cells obtained from thioglycolate-induced mice were washed with Hanks’ balanced salt solution (HBSS) and immediately used for further ex vivo experiments. For each experiment, leukocytes from 5 to 10 mice were pooled. January 2017 Volume 85 Issue 1 e00644-16

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Cell adhesion assays. Cell adhesion assays were performed as described previously (20, 34). Briefly, 48-well Costar tissue culture plates were coated in triplicate with 5 ␮g of the fibrinogen D fragment or the P2C peptide and postcoated with 0.5% polyvinylpyrrolidone. The selected isolated leukocyte populations (105 leukocytes) were added to each well and incubated for 30 min at 37°C. Subsequently, the plates were washed, and the number of adherent cells in each well was enumerated by using the CyQUANT cell proliferation assay kit (Becton Dickinson). Data are presented as percentages (means ⫾ standard errors [SE]) of total added cells (which were assigned a value of 100%) and represent the results of three independent experiments. Phagocytosis/intracellular killing assays. Thioglycolate-elicited PMN significantly deplete the contents of their primary and secondary granules during and immediately after transmigration into the peritoneal cavity. They are isolated mostly as degranulated phagocytic cells and then start to undergo apoptosis (74). Thioglycolate-elicited Δ␣M and Δ␣X PMN are defective in degranulation and capable of secreting only 60 to 70% of their granule content (37). However, the remainder of their granule content can be further released upon stimulation with a combination of agonists (21). Thus, the PMN recruited to the peritoneal cavity and stimulated with thioglycolate are able to eliminate pathogens only by intracellular killing mechanisms (37, 75, 76), while M␾ also eliminate pathogens by phagocytosis. The phagocytic/killing capabilities of PMN, iM␾, and rM␾ of all 3 mouse strains were determined as described previously (14, 21). Briefly, 105 C. albicans yeast cells were allowed to germinate in 0.25 ml RPMI 1640 at 37°C for 1 h to induce the expression of PRA1, the fungal ligand recognized by the two ␤2 integrins (14). Subsequently, fungi were washed and resuspended in 0.25 ml of HBSS containing 0.1 M HEPES (pH 7.4), 2 mM CaCl2, and 2 mM MgCl2 (HBSS-HEPES), and 7 ⫻ 105 (1:7 ratio) leukocytes were added. In some experiments, cells were preincubated with 5 ␮M PMA for 10 min at 22°C before addition to the fungus. E. coli cells (106) were used to determine LPS-induced phagocytosis (23). The leukocytefungus and leukocyte-bacterium mixtures were incubated at 37°C for 2 h. In some experiments, before the addition of the pathogens, leukocytes were preincubated with 20 ␮g/ml of blocking anti-mouse ␤2 MAb M18/2 or 2 ␮M ouabain (both from Sigma-Aldrich), which blocks the killing of phagocytized pathogens and their surface glycoproteins (39–41, 77) by interfering with phagolysosome formation and functions by blocking K⫹ transport (78). To determine the extent of intracellular killing, aliquots of the cell-pathogen suspensions were removed after 2 h, washed in HBSS-HEPES, lysed with 1% Tween 80, and plated in serial dilutions onto SDA and LB agar plates for C. albicans and E. coli, respectively. The CFU were counted manually by using a Bel-Art colony counter. The results are presented as percentages of surviving microbes, and samples containing only C. albicans or E. coli cells without added leukocytes were assigned a value of 100%. Pathogen-induced leukocyte recruitment into the peritoneal cavity. To determine the impact of ␣M and ␣X deficiencies on the initial antipathogen activity of mice in the context of the whole organism, we employed a modification of the murine peritonitis model used by us previously (33). Briefly, to initiate acute septicemia, WT, Δ␣M, and Δ␣X mice were challenged by intraperitoneal injection of either 106 C. albicans yeast cells or 107 E. coli cells (both inoculums are ⬎20-fold below the lethal doses). After 6 h or 72 h, the infected mice were euthanized, their peritonea were lavaged with cold PBS, cells in the lavage fluids were washed with HBSS and lysed with 1% Tween 80, and serial dilutions were plated in triplicate onto SDA (C. albicans) or LB agar (E. coli) plates for enumeration of surviving microbes as CFU. Murine model of fungal infection in presensitized peritoneal cavities. The murine model of fungal infection in presensitized peritoneal cavities is a modification of a two-stage model of intraperitoneal candidiasis (44). It involves the recruitment of leukocytes in response to thioglycolate with subsequent measurement of the antifungal activity of the recruited intraperitoneal cells. To commence the experiment, peritoneal cavities of WT, Δ␣M, and Δ␣X mice were injected with 0.5 ml of 4% thioglycolate in PBS. Six hours or 72 h after injection, mice were challenged i.p. with 106 C. albicans yeast cells. After 2 h, the mice were euthanized, and their peritoneal cavities were lavaged with cold PBS. Subsequently, cells in the lavage fluids were collected by centrifugation, washed, and lysed with 1% Tween 80, and the residual viable fungi were enumerated as CFU by plating series of dilutions onto SDA plates in triplicate. To control the ability of the pathogen to disseminate from the peritoneal cavity, CFU in the homogenates of retroperitoneal organs, kidney and duodenum, were determined. The antipathogen activity of PMN, iM␾, or rM␾ was determined by challenging the animals with C. albicans at the times of maximal recruitment into the peritoneal cavity after thioglycolate stimulation. Murine models of systemic candidiasis and cytokine secretion measurements. To analyze the impact of ␤2 integrin deficiency on protection against systemic fungal infection by residential M␾ in different tissues, we employed a comprehensive model described previously by MacCallum and Odds (48). This model is based on measuring fungal burdens in murine internal organs at selected time points to estimate fungal invasion and colonization (14). WT, Δ␣M, and Δ␣X mice (n ⫽ 5) were challenged intravenously with an inoculum of 105 C. albicans cells/mouse and were euthanized after 16 or 40 h of infection. Brains, livers, spleens, hearts, lungs, and kidneys were harvested, weighed, and homogenized. The homogenates were plated in serial dilutions onto SDA plates for CFU quantitation (48). Portions of the livers and kidneys were fixed separately in formalin and stained with anti-Ly6G or anti-F4/80 to identify leukocyte subpopulations infiltrating the organs (see below). For cytokine assays, mice (n ⫽ 5) were euthanized 24 h and 14 days (the predetermined endpoint of survival experiments) after initial challenge, and kidneys were removed and weighed. The levels of TNF-␣, IL-6, and IL-10 in the kidney homogenates were determined by using enzyme-linked immunosorbent assay (ELISA)-based commercial kits from Thermo Fisher Scientific (Waltham, MA) according to the manufacturer’s protocols. Model of acute endotoxemia. Mice (n ⫽ 10) of all 3 strains were injected intraperitoneally with purified endotoxin (LPS of E. coli serotype O111:B4; Sigma-Aldrich) at 5 mg/kg (79). Mouse survival was January 2017 Volume 85 Issue 1 e00644-16

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assessed with an endpoint scoring system as detailed previously (14, 21). In separate experiments, to estimate the possibility of organ specificity in the recruitment of leukocyte populations to inflamed organs, lungs of LPS-challenged mice were collected from euthanized animals immediately after injection (time zero) or at 48 h postinjection, sectioned, and subjected to immunocytochemical staining with antimacrophage F4/80 MAb (clone BM8; LifeSpan Bioscience, Seattle, WA). Histological analysis. Mouse tissues were collected at selected time points, fixed in 10% buffered formalin for 48 h, processed, and embedded in paraffin. Tissue sections (5 ␮m) were prepared by the use of an HM-340E microtome (Thermo Fisher) and placed onto glass slides (Fisher). Staining with hematoxylin and eosin (Fisher) followed by HRP-conjugated anti-murine Ly6G or anti-murine F4/80 (both from LifeSpan Bioscience, Seattle, WA) was performed. The numbers of F4/80- or Ly6G-positive cells were counted on at least eight random high-power fields (HPF) per slide, with 3 independent slides per point, and expressed as the mean number of cells/HPF ⫾ SE. Statistical analyses. Statistical significance was determined by using Student’s paired t test (from the statistical package in SigmaPlot, version 12.0; Jandel Scientific Software) and ANOVA (Jandel Scientific Software). Kaplan-Meier survival curves were analyzed by a log rank test. Differences between groups were considered significant at a P value of ⬍0.05. Data are expressed as means ⫾ standard deviations (SD) unless otherwise noted.

ACKNOWLEDGMENTS This work was supported by NIH grants AIO 80596 (National Institute of Allergy and Infectious Diseases, to D.A.S.) and PO1 HL073311 (National Heart, Lung, and Blood Institute, to E.F.P.). We thank Rajani Tendulkar and Nancy Fiordalisi for excellent administrative support of the project. We have no financial conflicts of interest.

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