Modulation of Inflammasome-Mediated Pulmonary Immune Activation by Type I IFNs Protects Homeostasis during Systemic Responses to Pneumocystis This information is current as Lung Infection of September 28, 2021. Steve Searles, Katherine Gauss, Michelle Wilkison, Teri R. Hoyt, Erin Dobrinen and Nicole Meissner J Immunol published online 23 August 2013 http://www.jimmunol.org/content/early/2013/08/23/jimmun Downloaded from ol.1301344 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 23, 2013, doi:10.4049/jimmunol.1301344 The Journal of Immunology

Modulation of Inflammasome-Mediated Pulmonary Immune Activation by Type I IFNs Protects Bone Marrow Homeostasis during Systemic Responses to Pneumocystis Lung Infection

Steve Searles,* Katherine Gauss,† Michelle Wilkison,‡ Teri R. Hoyt,‡ Erin Dobrinen,‡ and Nicole Meissner‡

Although acquired bone marrow failure (BMF) is considered a T cell–mediated autoimmune disease, possible innate immune defects as a cause for systemic immune deviations in response to otherwise innocuous infections have not been extensively explored. In this

regard, we recently demonstrated an important role of type I IFNs in protecting hematopoiesis during systemic stress responses to Downloaded from the opportunistic fungal pathogen Pneumocystis in lymphocyte-deficient mice. Mice deficient in both lymphocytes and type I IFN receptor (IFrag2/2 mice) develop rapidly progressing BMF due to accelerated bone marrow (BM) cell apoptosis associated with innate immune deviations in the BM in response to Pneumocystis lung infection. However, the communication pathway between lung and BM eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. In this study, we report that absence of an intact type I IFN system during Pneumocystis lung infection not only causes BMF in lymphocyte-deficient

mice but also transient BM stress in lymphocyte-competent mice. This is associated with an exuberant systemic IFN-g response. http://www.jimmunol.org/ IFN-g neutralization prevented Pneumocystis lung infection–induced BM depression in type I IFN receptor–deficient mice and prolonged survival time in BM from IFrag2/2 mice. IL-1b and upstream regulators of IFN-g, IL-12, and IL-18 were also upregulated in lung and serum of IFrag2/2 mice. In conjunction, there was exuberant inflammasome-mediated caspase-1 activation in pulmonary innate immune cells required for processing of IL-18 and IL-1b. Thus, absence of type I IFN signaling during Pneumocystis lung infection may result in deregulation of inflammasome-mediated pulmonary immune activation, causing systemic immune deviations triggering BMF in this model. The Journal of Immunology, 2013, 191: 000–000.

one marrow failure (BMF) can occur in the context of Pneumocystis is an extracellular, opportunistic fungal pathogen inherited and acquired conditions and manifests in its of the lung that causes a life-threatening pneumonia in severely by guest on September 28, 2021 B extreme form as aplastic anemia with severe peripheral immune compromised individuals with, for example, HIV infec- cytopenias and acellular BM spaces (1). Although most acquired tion or immunosuppressive therapy (9). Although the infection aplastic anemias are thought to be the result of a T cell–mediated often resolves unnoticed in otherwise healthy individuals, there autoimmune response to an unknown, likely infectious trigger, is increasing evidence that low-grade pulmonary Pneumocystis inherited forms are defined by gene defects often affecting the colonization/infection can exacerbate the symptoms of chronic pul- viability of hematopoietic stem cells (HSCs) in response to in- monary diseases such as chronic obstructive pulmonary disorder (10, flammatory stimuli (2–5). Furthermore, peripheral cytopenias due 11) and thus may also exacerbate systemic complications asso- to BM suppression can also occur as a complication of severe in- ciated with it (12). flammatory syndromes such as rheumatoid diseases, severe sepsis, As with many other fungal pathogens, immune protection from and AIDS (6–8). Thus, although pathomechanistically complex and Pneumocystis infection critically depends on CD4 T cell–medi- multifactorial, a common theme appears to be the presence of in- ated immune responses (13, 14). However, although immunity flammatory stimuli accompanied by immune deviations. to many other pulmonary fungal pathogens appears to involve inflammasome-mediated immune activation following innate pattern recognition and activation of a Th1/Th17-driven adaptive *Department of Pathology, University of California School of Medicine, La Jolla, CA immune response (15, 16), there is increasing evidence that suc- 92093; †Montana Veterinary Diagnostic Laboratory, Bozeman, MT 59718; and ‡Department of Immunology and Infectious Diseases, Montana State University, cessful immunity to Pneumocystis lung infection involves Th2- Bozeman, MT 59718 mediated immune responses including alternative Received for publication May 21, 2013. Accepted for publication July 22, 2013. activation and B cell–mediated clearance (17–22). This work was supported by National Institutes of Health (NIH) Grant RO1 Type I IFNs have long been known as antiviral (reviewed in Ref. HL90488 and NIH Centers of Biomedical Research Excellence (COBRE) Grant 23), and their role as mediators of immunity to bacterial and some 2P20RR020185. fungal infections has just been recognized (reviewed in Refs. 24, Address correspondence and reprint requests to Dr. Nicole Meissner, Department of 25). Type I IFNs activate , promote Immunology and Infectious Diseases, Montana State University, 960 Technology Boulevard, Bozeman, MT 59718. E-mail address: [email protected] (DC) maturation, enhance Th1- and NK cell–mediated immunity Abbreviations used in this article: BAL, bronchoalveolar lavage; BM, bone marrow; (26–28), but also support B cell differentiation to Ab-secreting BMF, bone marrow failure; DC, dendritic cell; HSC, ; plasma cells (29). Although type I IFN–mediated responses have 2/2 2/2 IFNAR , type I IFN receptor–deficient; IFrag , deficient in both lymphocytes been implicated in immune-mediated damage to specific patho- and type I IFN receptor; MHC II, MHC class II. gens (30) and autoimmune diseases (31, 32), they are also immune Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 modulators. In this regard, type I IFNs induce IL-10 production in

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301344 2 INFLAMMASOME-MEDIATED IMMUNE RESPONSES TO PNEUMOCYSTIS

LPS-stimulated macrophages (33) and in Ag-specific T cells, inflammasome-mediated immune activation in the lung in pre- leading to the suppression of a Th17-associated autoimmune in- venting systemic immune deviations and complications in this flammation in a mouse model of multiple sclerosis (34, 35). In model of Pneumocystis lung infection–induced BMF. addition, type I IFNs induce transcriptional repression of TNF-a (36), inhibition of inflammasome activation, and subsequent IL-1b processing (37) and are thus therapeutically used in patients with Materials and Methods multiple sclerosis (38–40) and evaluated for patients with chronic Mice inflammatory bowel diseases (41–43). All mice listed below were bred and maintained at Montana State University Therefore, type I IFNs are pleiotropic, and their activity is likely Animal Resource Center. Mice were housed in ventilator cages and received sterile food and water. C.B17 SCID mice, as a source for Pneumocystis dose dependent and determined by the immunological microen- 2/2 vironment. Indeed, low amounts of IFN-b accumulate in tissue in murina organism, and RAG1 mice (C57/BL6 background) were ini- tially purchased from The Jackson Laboratory (stock numbers 001803 and the absence of infection, maintaining a wide variety of signaling 002096, respectively). IFrag2/2 mice were generated by crossing IFNAR molecules important for immunity and tissue homeostasis (44). knockout mice (129SvEv/C57/BL6 background) with RAG12/2 mice Although in high dosages myelosuppressive (45, 46), type I IFNs (C57/BL6 background) as previously described (60) and have since been act as neutrophil survival factors similar to G-CSF (47). Further- backcrossed two more times on a C57/BL6 background. Lymphocyte- competent IFNAR2/2 mice initially used in experiments were on more, type I IFNs are critical regulators of bone homeostasis a 129SvEv/C57BL6 mixed background. However, subsequently all ex- (reviewed in Refs. 48, 49) and thus may also protect the bony HSC periments were repeated in IFNARnull/null mice, which are on a C57/BL6 niche and hematopoiesis (50, 51). However, excessive IFN-a background, and were kindly gifted to us by Dr. Ed Schmidt, Montana production during inflammatory responses can also induce and State University, and compared with C57/BL6 wild-type mice purchased at Downloaded from exhaust the proliferative capacity of the HSC and impact self- Charles River Laboratories (stock number 027). renewal (52). Pneumocystis lung infections and treatments The role of type I IFN responses during HIV infection has also Most experimental animals were intratracheally infected with 107 Pneu- been controversially discussed (53). Although early excessive type mocystis nuclei in 100 ml lung homogenate diluted in PBS buffer from I IFN production may contribute to immune activation and accel- infected source mice. Pneumocystis lung burden was assessed micro- erated loss of CD4 T cells (54), over the course of HIV infection, scopically by enumeration of trophozoid and cyst nuclei count in lung http://www.jimmunol.org/ CD4 T cells in conjunction with DCs, including type I IFN–pro- homogenates in 10–50 oil immersion fields as previously described (63). ducing plasmacytoid DCs, are sequentially deleted and/or their The limit of detection for this technique is log10 4.43. For some experi- ments, mice received limiting dilutions of 105 and 103 nuclei. Some functions are impaired (55–57). The loss of these cell types con- control groups of mice received an inoculum of 107 heat-inactivated nuclei tributes together and independently to the progression of immune or clean lung homogenate from uninfected CB17 source mice. Anti–IFN-g deregulation and immunodeficiency in HIV+ patients (58, 59). treatment was performed by i.p. injection of purified 250 mg anti–IFN-g We recently evaluated the role of type I IFNs during Pneumo- Ab (clone R4-6A2; American Type Culture Collection; three times per week). Some mice were placed on antibiotic medicated food (Sulfatrim cystis lung infection in lymphocyte-competent and lymphocyte- rodent pellets; LabDiet) Mice were euthanized by exsanguination fol- deficient mice and also observed type I IFN–mediated pleiotropic lowing induction of deep pentobarbital narcosis. All mouse studies con- effects on local and systemic immune responses to the infection. formed to National Institutes of Health guidelines and were approved by by guest on September 28, 2021 In a CD4 T cell–depleted mouse model of Pneumocystis pneu- the Institutional Animal Care and Use Committee at Montana State Uni- monia, type I IFNs promote CD8 T cell–induced lung damage. versity. However, absence of type I IFN signaling in lymphocyte-competent Cryptococcus lung infection and evaluation of lung burden mice (type I IFN receptor–deficient [IFNAR2/2]) induces prolonged In some experiments, mice were intratracheally infected with 5 3 103 pulmonary inflammation despite pathogen clearance eliciting fibrosis Cryptococcus neoformans yeast (strain H99; kindly provided to us by (18). In addition, we found that type I IFN signaling plays an es- Dr. Robert Cramer, Montana State University) in 100 ml PBS. For this, yeast sential role in maintaining hematopoiesis during the systemic acute was streaked from frozen stocks onto YPD agar plates and incubated at phase response to this fungal infection (60). Combined lymphocyte- 37˚C for 48 h. To generate a liquid culture, a single colony was picked and and IFNAR-deficient mice (IFrag2/2), but not lymphocyte-deficient inoculated into 10 ml YPD media and further incubated at 37˚C. For in- 2/2 fection, yeast were enumerated, washed, and adjusted to the appropriate but IFNAR-competent mice (RAG ), develop rapidly pro- concentration in sterile PBS. Infected mice were euthanized at 7 and gressing BMF in response to Pneumocystis lung infection. This is 10 d postinfection, the right lung removed, homogenized in 5 ml PBS, and due to accelerated apoptosis of maturing combined 100 ml were plated onto YPD agar plates at 1:10, 1:100, and 1:1000 with lack of replenishment from early progenitors (61). Induction dilutions in PBS, incubated for 3 d, and colonies counted. 2/2 of BMF in IFrag mice is associated with the deregulated ex- Collection and differentiation of BM cells pression of proinflammatory and proapoptotic cytokines (e.g., TNF-a, IL-1, and TRAIL) and exuberant reactive oxygen species BM cells from femur and tibia were collected as previously described by flushing 2 ml PBS through the BM canal using a 26.5-gauge needle and induction in the BM (61, 62). However, the communication brought into a single-cell suspension (64). pathway initiated between lung and BM resulting in the induction BM cells were diluted 1:10 in PBS. Cell numbers were enumerated, spun of BMF in response to this strictly pulmonary infection has been onto glass slides, and stained with Diff-Quick solution (Dade Behring). Cell unclear. differentiation was performed based on morphology and staining pattern to distinguish myeloid (including – and metamyelocyte In this study, we report that absence of an intact type I IFN system stage), banded versus segmented , , and others (in- elicits an innate and exuberant systemic IFN-g (type II IFN) response cluding erythroid, megakaryocytic, and lymphoid cells) (64). to Pneumocystis lung infection that contributes to induction of BM stress in both IFNAR2/2 and IFrag2/2 mice, respectively. IFN-g– FACS analysis of cellular subsets in BM and lung inducing cytokines such as IL-12 and IL-18, in concert with IL-1b, FACS analysis was also applied to lung and BM cells, combining cell- were concomitantly upregulated in lung and serum of IFrag2/2 mice surface staining in combination with assessment for caspase-1 activities and associated with exuberant inflammasome-mediated caspase-1 at the single-cell level or expression of intracellular IFN-g. Prior to FACS staining, RBC lysis of BM and lung cell samples was performed using activation in pulmonary innate immune cells in response to Pneu- 3 7 2/2 ACK lysis buffer. Cells were then suspended at 1 10 /ml in FACS buffer mocystis lung infection when compared with RAG mice. Our (PBS/10% calf serum) containing Fc-block (mouse clone 24G2; BD data point to an important role of type I IFNs in regulating of Pharmingen) at a 1:800 dilution. Sets of 5 3 105 cells were stained with The Journal of Immunology 3 specific Abs: MHC class II (MHC II; PerCP–Cy5-5, clone M5/114.15.2; Results eBioscience), anti-CD11b (Alexa Fluor 700; clone M1/70; BioLegend), Lymphocyte-competent IFNAR2/2 mice develop a transient anti–Ly-6G/6C (allophycocyanin-Cy7; clone RB6-8C5; BD Pharmingen), anti-NK1.1 (PE-Cy7, clone PK136; BD Pharmingen), anti-CD11c (allo- BM crisis in response to Pneumocystis lung infection phycocyanin, clone N418; eBioscience), anti–IFN-g (V450, clone XMG1.2; Complete BMF develops in IFrag2/2 but not RAG2/2 mice in re- BD Biosciences), or FAM-FLICA Caspase-1 (Immunochemistry Technolo- gies) according to the manufacturer’s instructions. To obtain single-cell sponse to Pneumocystis lung infection within 16–21 d postinfection. suspensions of lung cells for FACS analysis of lung cells, right lung lobes It is reflected by loss of total BM numbers and hematopoietic were removed, diced with scissors, placed into 10 ml PBS containing 1 mg/ progenitor/stem cell activity (CFU count) (Fig. 1A) and subsequently ml Collagenase D (Roche), and incubated for 30 min at 37˚C. Lungs were leads to premature death of the animal. In contrast, lymphocyte-com then placed into a 100-mm mesh filter screen and carefully pushed through petent IFNAR2/2 mice appear clinically unimpaired, although previ- into a clean tube. For the evaluation of IFN-g production of cellular subsets, cells were incubated for 4 h in the presence of 40 ng/ml PMA and 1 mg/ml ously observed significant splenomegaly suggested extramedullary he- ionomycin in MEM in the presence of 1 mg/ml brefeldin A. Following cell- matopoiesis (60). surface staining with the above-mentioned surface markers, cells were fixed We now have found that IFNAR2/2 mice (not wild-type mice) and permeabilized using Cytofix/Cytoperm reagents from BD Biosciences also develop significant but transient BM depression in response to following the manufacturer’s protocol and stained with either 0.5 mge450- labeled isotype-control Ab or anti–IFN-g. Following repeated wash steps, Pneumocystis lung infection, which is maximal at day 10 post- samples were acquired using an LSR BD FACS machine (BD Biosciences) infection (Fig. 1B). This was largely due to loss of neutrophils and 2 2 and analyzed using FlowJo Software (Tree Star). thus mirrored the early phenotype previously observed in IFrag / mice (61). In contrast to IFrag2/2 mice, IFNAR2/2 mice re- Lung cell analysis tained significant hematopoietic progenitor cell activity within Downloaded from Bronchoalveolar lavages (BALs) were performed as previously described the BM, which was reflected by high CFU counts of plated BM (65). Briefly, mice were i.p. injected with 90 mg/kg sodium pentobarbital cells in vitro. In fact, BM CFU counts in IFNAR2/2 mice in- solution and exsanguinated. BALs were performed by intratracheal can- nulation, and instillation and extraction of 5 3 1 ml HBSS containing creased when BM depression was maximal. However, consistent 2/2 3 mM EDTA. Lung lavage cells were enumerated using a hemocytometer, with evidence of BM stress, and different from IFrag mice, spun onto glass slides, and stained with Diff-Quick (Siemens) for differ- extramedullary hematopoiesis in the spleen was suggested by in- ential counting. FACS analysis was performed as described above to eval- creased spleen size and could be confirmed by increased hema- uate caspase-1 activity as indicator of inflammasome activation in cellular http://www.jimmunol.org/ subsets. topoietic progenitor activity when were placed in CFU assays (Fig. 1C). Furthermore, and consistent with BM de- Serum samples pression, IFNAR2/2 mice demonstrated a deviated, infection- Serum samples were collected throughout the course of infection by har- induced BM cytokine profile previously shown to be associated 2 2 vesting full samples at each time point, which were spun and the buffy with BMF in IFrag / mice (62). This included a transient up- coat separated from the serum. Samples were frozen until further analysis. regulation of proinflammatory and proapoptotic cytokines such as Cytokine analysis TNF-a, IL-1b, and TRAIL (Fig. 2A–C). Cytokine analysis from BAL, serum, and BM cell lysate was performed Induction of BMF by Pneumocystis lung infection is dependent by guest on September 28, 2021 using multiplex assay plates from either Multiplex or Bio-Rad Life Sciences on the infectious dose and requires live pathogen using a Bioplex (Bio-Rad) analysis system. BM cell lysates were generated from a cell concentration of 2.5 3 107/ml using cell lysis buffer from Pneumocystis does not appear to disseminate from the lung to the BioVison. The following cytokines were evaluated: IFN-g, IL-1b, IL-18, BM in our model (60). Thus, we hypothesized that deviated cy- IL-12p70, IL-5, IL-10, IL-15, and TNF-a using this methodology. In some 2/2 2/2 experiments, IFN-g protein concentrations were separately measured using tokine responses observed in the BM of IFrag and IFNAR ELISA reagents from R&D Systems (Mouse IFNg Duoset). Furthermore, mice in response to Pneumocystis lung infection and associated in some experiments, TRAIL concentrations were also evaluated in BM with BM pathology were caused by actions of immune mediators cell lysates using ELISA reagents from R&D Systems (Mouse TRAIL released from the lung into the system. Duoset). Therefore, we first evaluated whether Pneumocystis-induced Colony-forming cell assay for mouse BM cells systemic immune deviations are dependent on the infectious dose

2/2 2/2 and whether it required live pathogen. For this, we evaluated total Hematopoietic precursor cell activity in BM from IFrag and RAG 2/2 mice was assessed by hematopoietic colony-forming counts in methylcel- BM cell numbers and composition of six groups of IFrag mice lulose media as previously described (61). For this, 105 BM cells/animal at day 16 postinfection that had received a variety of P. murina and group at each time point were plated in MethCult GF M3534 media dosages: 1) standard dosage of 107 live Pneumocystis nuclei; 2) (StemCell Technologies), which has been formulated to support the optimal 105 live Pneumocystis nuclei; 3) 103 live Pneumocystis nuclei; 4) growth of and macrophage precursor cells. Cells from each 7 sample were plated in duplicate according to the manufacturer’s protocol in 10 nuclei of heat-inactivated Pneumocystis; 5) clean lung ho- 35-mm sterile culture dishes (StemCell Technologies) and placed in 100- mogenate from an uninfected SCID source mice; and 6) unin- mm Petri dishes in the presence of one 35-mm dish containing sterile water. fected and unmanipulated. Cultures were incubated for 7 d in a water-jacketed incubator maintained Fig. 3A shows Pneumocystis lung burden at day 16 for the with 5% CO2. Colony recognition (GM-, G-, and M-CSF) and enumeration respective experimental group, demonstrating lower pathogen load were performed according to StemCell Technologies’ guidelines. in those mice receiving lower infectious dosages and no Pneu- Microscopy mocystis growth in those receiving heat-inactivated pathogen or Microscopic evaluations of cytospin slides were performed using a Nikon clean lung homogenate. Fig. 3B shows respective BM cell num- Eclipse E200 microscope (Nikon) at 31000 original magnification under bers. BMF could still be induced at the same rate of 16 d with a oil immersion. BM colony count analysis (hematopoietic colony-forming 100-fold reduced Pneumocystis dosage of 105 nuclei. In contrast, count assays) was performed using a Zeiss Axiovert microscope (Carl inoculation with 103 live nuclei, as well as inoculation with heat- Zeiss) under 325 and 350 original magnification. inactivated pathogen (107 dead nuclei) and clean lung homoge- Statistical analysis nate, left the BM unaffected within the observed time frame. 2/2 Data were plotted using Prism GraphPad Software (GraphPad), and sta- Interestingly, initiation of antibiotic treatment of IFrag mice tistical analysis was performed using either a one-way or two-way ANOVA, with sulfamethoxazole/trimethoprim chow either beginning prior followed by a Tukey or Bonferroni posthoc test, respectively. to (day 23) or at days 0, 3, or 7 postinoculation with 107 (or 105) 4 INFLAMMASOME-MEDIATED IMMUNE RESPONSES TO PNEUMOCYSTIS Downloaded from http://www.jimmunol.org/

FIGURE 1. Type I IFN signaling is critical in regulating hematopoiesis during systemic responses to Pneumocystis lung infection in both lymphocyte- deficient and lymphocyte-competent mice. (A) Shown are total BM cell numbers, hematopoietic colony-forming activity (CFU counts) of BM cells, and P. murina (PC) lung burden from IFrag2/2 (IFNAR2/2/RAG2/2) and RAG2/2 mice (IFNAR+/+/RAG2/2) over the course of Pneumocystis lung infection at days 0, 7, 10, and 16 postinfection. (B) Comparative analysis of total BM cell numbers, hematopoietic colony-forming activity (CFU counts) of BM cells and PC lung burden from lymphocyte-competent IFNAR2/2 (IFNAR2/2/RAG+/+) mice and wild-type (WT) mice (IFNAR+/+/RAG+/+) over the course of Pneumocystis lung infection at days 0, 7, 10, and 16 postinfection. (C) Demonstrated are total spleen counts and hematopoietic colony-forming activity (CFU counts) of spleen cells from IFNAR2/2 and WT mice as a measure for extramedullary hematopoiesis at days 0, 7, 10, and 16 postinfection. Statistical by guest on September 28, 2021 analysis was performed using a two-way ANOVA. Comparisons were made between groups at the same time point. *p , 0.05, **p , 0.01, ***p , 0.001.

P. murina nuclei could not prevent progression of BMF, despite mice in response to Pneumocystis lung infection that could be subsequent suppression of P. murina burden near detection limit at linked to the induction of BM stress. day 16 postinfection (Fig. 3C, 3D). IFN-g, a type II IFN cytokine, is produced by an array of innate

2/2 2/2 2/2 and adaptive immune cells (66, 67) and has been implicated in IFNAR and IFrag , but not wild-type and RAG the pathogenesis of BMF in other models of the disease (68, 69). mice, demonstrate an exuberant serum IFN-g response to Thus, we compared IFN-g serum levels during early responses Pneumocystis lung infection that is associated with to Pneumocystis lung infection in IFrag2/2 and RAG2/2 mice as induction of BM pathology well as in IFNAR2/2 and wild-type mice over the course of in- As previously published, Pneumocystis lung infection elicits a fection and found significantly higher IFN-g serum levels in both strong but transient systemic type I IFN response with elevated IFNAR2/2 and in IFrag2/2 mice, when compared with wild-type serum IFN-a levels in wild-type and RAG2/2 mice (62). In this and RAG2/2 mice (Fig. 4A, 4B). Although in some experiments, study, we further evaluated whether specific immune mediators IFN-g serum responses appeared transient, peaking at day 7, in were elicited systemically and uniquely in IFrag2/2 and IFNAR2/2 other experiments, IFN-g levels remained high following induc-

FIGURE 2. Transient BM depression during Pneumocystis lung infection in IFNAR2/2 mice is associated with deviated cytokine responses as previously seen in IFrag2/2 mice during progression of BMF. Comparative cytokine analysis was performed in BM lysates isolated from IFNAR2/2 and wild-type mice at day 0, 7, 10, and 16 post–Pneumocystis lung infection using an ELISA-based multiplex assay system. Shown are data for TNF-a (A), IL-1b (B), and TRAIL (C). Statistical analysis was performed using a two-way ANOVA. Comparisons were made between groups at the same time point. ***p , 0.001. The Journal of Immunology 5

FIGURE 3. Induction of BMF in lymphocyte- deficient IFrag2/2 mice depends on the exposure to a critical dose of 105 live pathogen nuclei and cannot be prevented by antibiotic treatment. Groups of IFrag2/2 mice were intratracheally inoculated with 107,105, and 103 live Pneumocystis (PC) nu- clei or 107 dead PC nuclei and BM responses compared with animals either inoculated with clean lung homogenate (LH) at day 16 postinfection and uninfected animals. (A) shows PC lung burden at day 16 postinfection following microscopic enu- meration. (B) shows total BM counts of all IFrag2/2 comparison groups at day 16 postinfection. Anti- biotic treatment was initiated in groups of IFrag2/2 mice 3 d prior to (day 23) and at days 0, 3, and 7 Downloaded from postinoculation with 107 PC nuclei. BM cell num- bers and PC lung burden were evaluated and com- pared with uninfected mice at day 16 postinfection. (C) shows PC lung burden, and (D) shows total BM counts at day 16 postinfection. Statistical analysis was performed using a one-way ANOVA. All groups were compared with those mice receiving http://www.jimmunol.org/ 107 PC nuclei. *p , 0.05, **p , 0.01, ***p , 0.001. SMX/TMP, Sulfamethoxazole/trimethoprim.

tion throughout day 16 postinfection (see also Fig. 8). Regardless, Cryptococcus over a course of 10 d. Indeed, we found that IFN-g induction of high serum IFN-g levels coincided with or preceded serum levels were uniquely elevated in response to Pneumocystis, by guest on September 28, 2021 the induction of BM depression/BMF, respectively. but not to Cryptococcus, lung infection (Fig. 5B), although the The induction of BMF in IFrag2/2 mice appears specific to pathogen lung burden for both Pneumocystis and Cryptococcus responses to Pneumocystis lung infection (60). C. neoformans (Fig. 5C) continuously increased in both animal groups over the (strain H99) does not induce BMF in IFrag2/2 mice (Fig. 5A), tested time period. although mice succumb to the infection within 21 d if intra- Anti–IFN-g treatment prevented BM depression in IFNAR2/2 3 3 tracheally infected with a low infectious dose of 5 10 yeast cells. mice and ameliorated progression of BMF in IFrag2/2 mice Thus, we also asked if high serum IFN-g levels were also unique to responses Pneumocystis lung infection in our model and compared To determine if a cause-and-effect relationship existed between serum IFN-g levels of IFrag2/2 mice infected with Pneumocystis or elevated systemic IFN-g levels and BM pathology in our systems, we first treated lymphocyte-competent IFNAR2/2 mice with neutralizing anti–IFN-g Ab (clone R4-6A2) and compared their BM response to those that were left untreated over a time course of 16 d post–Pneumocystis lung infection. As demonstrated in Fig. 6, treatment of Pneumocystis-infected IFNAR2/2 mice averted the transient BM crisis normally present at day 10 postinfection (Fig. 6A) by preventing accelerated loss of band and segmented neu- trophils (Fig. 6B, 6C). Amelioration of BM stress was associated with reduced extramedullary hematopoiesis (Fig. 6D) and specific reduction in CFU granulocyte formation as a measure of extra- medullary (Fig.6E).Furthermore,anti–IFN-g FIGURE 4. IFNAR2/2 and IFrag2/2 show elevated serum IFN-g levels treatment reduced upregulation of TRAIL receptor DR-5 (Fig. 6F) during early responses to Pneumocystis lung infection, which correlates and TRAIL expression in the BM (Fig. 6G) normally associated with induction of BM stress. IFN-g serum evaluated over the course of with BM depression in Pneumocystis-infected IFNAR2/2 mice Pneumocystis lung infection in IFNAR2/2, IFrag2/2, wild-type, and 2/2 (see Fig. 2C). RAG mice at days 0, 7, 10, and 16 postinfection using a plate-based Despite the profound effects of anti–IFN-g treatment on he- ELISA assay. (A) compares serum IFN-g responses between lymphocyte- 2/2 2 2 matopoiesis in lymphocyte-competent IFNAR mice, the same competent IFNAR / and wild-type mice. (B) compares serum IFN-g responses between lymphocyte-competent IFrag2/2 and RAG2/2 mice. treatment had only a partial effect when performed in Pneumo- 2/2 2/2 Statistical analysis was performed using a two-way ANOVA. Comparisons cystis-infected, lymphocyte-deficient IFrag mice. In IFrag were made between groups at the same time point. **p , 0.01, ***p , mice, anti–IFN-g treatment ameliorated the progression of BMF 0.001. (Fig. 7A), seemingly by prolonging the survival time of segmented 6 INFLAMMASOME-MEDIATED IMMUNE RESPONSES TO PNEUMOCYSTIS

concentrations at day 10 postinfection (Fig. 8A–D), whereas IL-15 was not differentially regulated. Surprisingly, the immunosup- pressive cytokine IL-10 was also uniquely increased in serum IFrag2/2 mice despite high levels of IFN-g and other proinflam- matory cytokines. IL-12 and IL-18, as well as IL-1b and IFN-g, can be produced by a variety of innate immune cells, including macrophages and DCs following TLR-mediated activation (70, 71). To assess whether the source of elevated serum cytokines was primarily located in the lung, we measured BAL concentrations for IL-12, IL-18, IL-1b, and IFN-g in BAL fluid from IFrag2/2 and RAG2/2 mice at days 6 and 10 postinfection. Although we found uniquely elevated levels for IL-12p70, IL-18, and IL-1b in BAL fluid of IFrag2/2 mice in response to Pneumocystis lung infection at days 6 and 10 postinfection (Fig. 9A–C), we were repeatedly unable to detect elevated IFN-g in the BALs of IFrag2/2 mice (data not shown), suggesting either an interstitially located cellular source or a source located outside of the lung. Downloaded from Deviated local and systemic immune activation to Pneumocystis lung infection in IFrag2/2 mice is associated with increased inflammasome-mediated caspase-1 activity in the lung Although IL-12p70 is directly secreted as a heterodimer from its source (72), IL-18 and IL-1b require inflammasome-activated

caspase-1 processing to be released in its bioactive form (73– http://www.jimmunol.org/ 75). To evaluate if elevated local and systemic cytokine levels in IFrag2/2 mice were associated with increased inflammasome- mediated immune activation in the lung, we assessed caspase-1 activity in cells from BAL fluid and lung digests of IFrag2/2 compared with RAG2/2 mice at days 0, 7, and 10 post–Pneu- FIGURE 5. C. neoformans lung infection does not induce BMF in 2 2 mocystis lung infection using a FACS-based fluorescent assay IFrag / mice and also does not induce a systemic IFN-g serum response. (A) compares total BM cell numbers of IFrag2/2 mice either infected with (FAM-FLICA; Immunochemistry Technologies) in conjunction C. neoformans (Crypto; strain H99) or Pneumocystis (PC) at days 0, 7, 10, with cell-surface marker staining. As demonstrated in Fig. 10, we + and 16 postinfection. (B) compares serum IFN-g levels between Crypto found a significantly higher percentage of active caspase-1 cells by guest on September 28, 2021 2 2 and PC-infected IFrag2/2 mice at days 0, 7, and 10 postinfection. Statis- in BAL fluid (Fig. 10A) and lung digest (Fig. 10B) of IFrag / 2 2 tical analysis was performed using a two-way ANOVA. Comparisons were mice when compared with RAG / mice in a kinetic consistent made between groups at the same time point. (C) shows the respective with high BAL and serum IL-18 and IL-1b concentrations (Figs. pulmonary fungal burden for PC (left axis) and Crypto (right axis) over the 8D–F, 9B, 9C) and induction of BMF. The majority of caspase-1+ respective time course. Statistical analysis was performed using a one-way cells in the BAL were CD11c+ MHC II+ cells macrophage-type , ANOVA comparing fungal burdens within the respective groups. *p cells and CD11b+Gr-1+ neutrophils in lung digest (data no shown). 0.05, **p , 0.01, ***p , 0.001. These data implicate exuberant inflammasome-mediated immune activation in pulmonary innate immune cells as a contributor to and band neutrophils in the BM (Fig. 7B, 7C). However, neu- 2/2 trophils subsequently disappeared and remained unreplenished, the systemic immune deregulation in IFrag mice. similar to what was seen in untreated IFrag2/2 mice. This sug- gested an additional mechanism responsible for the full progres- Discussion sion of the disease in lymphocyte-deficient mice. We recently found type I IFNs to be critical in supporting on- demand hematopoiesis during systemic acute-phase responses to Induction of a systemic IFN-g response to Pneumocystis lung 2/2 Pneumocystis lung infection, especially in lymphocyte-deficient infection in IFrag mice is associated with increased serum mice (60, 61). In the current study, we further evaluated the sys- and BAL levels of IL-18 and IL-12 temic communication pathway between lung and BM and how Neutralization experiments suggested a partial role of systemically type I IFNs modulate early innate pulmonary responses to prevent upregulated IFN-g in the pathogenesis of BMF in IFrag2/2 mice. the induction of BMF in our model. However, understanding mechanisms causing its deregulation Although we previously showed that lymphocyte-competent could lend insight into the global mechanisms by which type I IFNAR2/2 mice are protected from the progression to complete IFNs act to prevent systemic immune deviation associated with BMF by yet unclear mechanisms provided by B cells (60), we now BMF in response to Pneumocystis lung infection. Therefore, we know that lymphocyte-competent IFNAR2/2 mice nevertheless assessed serum IFN-g responses of IFrag2/2 compared with develop a transient BM crisis with significant loss of BM neu- RAG2/2 mice over the course of Pneumocystis lung infection trophils. This crisis is associated with similar BM cytokine devia- (days 0, 7, 10, and 16 postinfection) in conjunction with an array tions previously noted in IFrag2/2 mice (62) that are consistent of cytokines that are known to induce or regulate IFN-g produc- with neutrophil apoptosis (76, 77) and include upregulation tion (IL-12p70, IL-15, IL-18, IL-1b, and IL-10) (67). As shown of TNF-a and TRAIL. However, in contrast to IFrag2/2 in Fig. 8, serum analysis demonstrated increased levels of IFN-g mice, lymphocyte-competent IFNAR2/2 mice retain hematopoi- in conjunction with IL-12, IL-18, and also IL-1b,uniquelyin etic progenitor activity with the ability to replenish transiently lost samples from IFrag2/2 mice starting at day 7, with maximum cells, although significant extramedullary hematopoiesis serves The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. Anti–IFN-g treatment prevents the transient BM crisis in lymphocyte- competent IFNAR2/2 mice. IFNAR2/2 mice were infected with Pneumocystis and received either neutralizing anti–IFN-g (three times weekly 250 mg; clone R4-6A2) or remained untreated. BM and spleen responses were analyzed at days 0, 10, and 16 postinfection. Shown in (A)–(C), bare comparative analyses of total BM cell numbers (A) and BM cell differentiation with percentage of band neutrophils (B) and segmented neutrophils (C) between treated and untreated IFNAR2/2 mice. (D) shows comparative total CFU counts of spleen cells as a measure of extramedullary hematopoiesis. (E) shows the percentage of CFU granulocytes (CFU-G) within total CFUs counted as a measure for extramedullary granulopoiesis. (F) shows the percentage of TRAIL-receptor (DR5+) BM cells plotted as percent positive cells over the course by guest on September 28, 2021 of infection. (G) shows TRAIL protein levels in BM cell lysates of comparison groups over the course of infection. Statistical analysis was performed using a two-way ANOVA. Comparisons were made between the groups at the same time point. **p , 0.01, ***p , 0.001. as additional evidence of BM stress. These distinct findings between istered compared with the initial infection strategy, but did not lymphocyte-competent IFNAR2/2 and lymphocyte-deficient develop within the time frame of 16 d when a 10,000-fold reduced IFrag2/2 mice suggest a two-hit process in the pathogenesis of inoculum was used. Surprisingly, antibiotic treatment with BMF in our model. A first hit that affects survival time of mature sulfamethoxazole/trimethoprim prior to or at an early time point and maturing neutrophils, whereas a second hit affects the health following infection with 105 or 107 P. murina nuclei could not of early progenitors/HSCs, resulting in lack of replenishment. prevent the progression of BMF in IFrag2/2 mice. This experi- Although possible, Pneumocystis has rarely been shown to ment suggested that a specific level of pathogen inoculum is re- disseminate to other organ sites, especially to the BM (78, 79). We quired to reach a threshold of immunological detection that, when show in this study that in IFrag2/2 mice, BMF still progresses surpassed, in the absence of type I IFN signaling, sets off a series unchanged when 100-fold less pathogen (105 nuclei) was admin- of responses that initiate BMF in our model.

FIGURE 7. Anti–IFN-g treatment ameliorates loss of mature neutrophils but cannot prevent the progression of BMF in lymphocyte-deficient IFrag2/2 mice. IFrag2/2 mice were infected with Pneumocystis and received either neutralizing anti–IFN-g (three times weekly 250 mg; clone R4-6A2) or no treatment. Total BM cell numbers and cell differentiation were performed and compared between the treated (open circle) and untreated group (closed circle) at days 0, 7, 10, and 16 postinfection. (A) shows total BM cell numbers. (B) and (C) show the percentage of band and segmented neutrophils in the BM of the comparison groups. Statistical analysis was performed using a two-way ANOVA. Comparisons between the groups were made at each time point. *p , 0.05. 8 INFLAMMASOME-MEDIATED IMMUNE RESPONSES TO PNEUMOCYSTIS

FIGURE 8. Cytokines considered inducers and reg- ulators of IFN-g are concomitantly upregulated in the serum of IFrag2/2 but not RAG2/2 mice in response to Pneumocystis lung infection. Comparative serum cyto- kine analysis was performed on IFrag2/2 and RAG2/2 mice at days 0, 7, 10, and 16 post–Pneumocystis lung infection using a multiplex ELISA-based assay to evaluate if other proinflammatory and IFN-g regulatory cytokines are uniquely induced in IFrag2/2 mice.

Shown are comparative data for IFN-g (A), IL-12(p70) Downloaded from (B), IL-18 (C), IL-1b (D), IL-15 (E), and IL-10 (F). Statistical analysis was performed using a two-way ANOVA. Comparisons reflect differences between the groups at each individual time point. *p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/ by guest on September 28, 2021

Pneumocystis, like a variety of other fungal pathogens, is rec- b-glucans from microbe-bound b-glucans, and dectin-1-signaling ognized by innate immune cells via engagement of dectin-1 re- is only activated by microbe-bound particulate and not by soluble ceptor with fungal cell wall b-glucans (80–82), which sets off b-glucans. Therefore, we concluded that abnormal immune respon- a strong innate inflammatory program. Although we never had ses in the lung, induced in the absence of type I IFN signaling spe- evidence that Pneumocystis disseminates from the lung to the BM cifically in response to Pneumocystis infection, must cause systemic in our system, in some experiments, we were able to detect soluble immune deviations that initiate aberrant BM responses in IFrag2/2 fungal b-glucans in the serum of IFrag2/2 mice around days 5 to 6 and IFNAR2/2 mice, leading to BMF or stress, respectively. postinfection (data not shown). This was of interest, as C. neo- Responses to IFN-g (and TNF-a) have been implicated in the formans, due to its capsule, does not shed b-glucans systemically pathogenesis of acquired and some inherited BMF syndromes (2, (83) or induces BMF in our system. Although this could be a 68, 69, 85–88), and cross-regulation between these cytokines and mechanism by which this strictly pulmonary infection elicits type I IFNs exists (26, 89, 90). We previously demonstrated that significant systemic effects, recent work by Goodridge et al. (84) absence of type I IFN signaling results in an early exuberant, demonstrated that innate immune cells distinguish soluble pulmonary TNF-a and IFN-g response in IFNAR2/2 mice (91).

FIGURE 9. Cytokine profile of BAL fluid (BALF) in part mirrors serum cytokine profile. Comparative cytokine analysis of BALF from IFrag2/2 and RAG2/2 mice with focus on proinflammatory cytokines and upstream regulators of IFN-g using a multiplex assay system. Demonstrated are comparative results for IL-12p70 (A), IL-18 (B), and IL-1b (C). Statistical analysis was performed using a one-way ANOVA. Comparisons were made between the groups at each time point. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 9

FIGURE 10. Increased BAL and serum IL-18 and IL-1b levels in IFrag2/2 mice are associated with increased induction of inflammasome-mediated caspase- 1 activity in cellular sources of BAL and lung digest. Caspase-1 activity was evaluated on a single-cell level in cellular sources of BAL fluid and lung digest of IFrag2/2 and RAG2/2 mice at days 0, 7, 10, and 16 of Pneumocystis lung infection using a live cell assay in which fluorescently labeled caspase-1 inhibitor (FAM-FLICA) enters the cell and binds irreversibly to activated caspase-1, allowing subsequent FACS analysis in combination with cell surface marker staining. (A) shows the percentage of active caspase-1+ cells in BAL fluid, and (B) shows the percentage of caspase-1+ cells in lung digest single-cell suspensions. Statistical analysis was performed using a two-way ANOVA. Comparisons were made between groups at each time point. *p , 0.05, ***p , 0.001. Downloaded from Indeed, in this study, we also found high IFN-g serum concen- monary deregulation in IFNAR-deficient mice ultimately respon- trations uniquely in IFNAR2/2 and IFrag2/2 mice, but not in sible for initiation of all systemic immune deviations contributing wild-type and RAG2/2 mice specifically induced in response to to the induction (first hit) and progression (second hit) of BMF in Pneumocystis lung infection. Importantly, the induction of high our model. serum IFN-g levels between days 7 and 10 coincided with the IL-18 in combination with IL-12 are potent inducers of IFN-g induction of BM stress in these mice and was not induced when (96–99), whereas IL-10 is considered an important negative reg- http://www.jimmunol.org/ mice were infected with C. neoformans. ulator of IFN-g transcription (100). In a recent study, we found IFN-g is a powerful proinflammatory mediator produced by an high IFN-g protein levels in BAL fluid, in conjunction with array of innate and adaptive immune cells that coordinates a di- a significant upregulation of IL-18 and IL-12 mRNA in pulmonary verse array of cellular programs via transcriptional regulation, CD11c+ cells uniquely from lymphocyte-competent IFNAR2/2 including pro- and antiapoptotic pathways (66). Although both mice, but not wild-type mice, during early time points of Pneu- type I IFNs and IFN-g prolong neutrophil survival time at in- mocystis lung infection (days 7 and 14, respectively) (91). This flammatory sites via the induction of antiapoptotic regulators (47), suggested a distinctive pulmonary immune activation pathway in IFN-g also promotes apoptosis (e.g., via induction of TRAIL ex- response to Pneumocystis lung infection in the absence of type pression in a variety of tissue types) (92–95). The regulation of I IFN signaling. When these cytokines were evaluated in by guest on September 28, 2021 TRAIL by IFN-g is of interest as TRAIL may provide the first hit lymphocyte-deficient mice, we also found elevated IL-18 and in the progression of BMF in our model. Indeed, TRAIL has been IL-12 in conjunction with IL-1b protein levels uniquely present in shown to accelerate neutrophil apoptosis and aid in eliminating the serum of IFrag2/2 mice, and not in RAG2/2 mice, which also senescent neutrophils form the BM (76). Furthermore, we previ- coincided with elevated serum IFN-g during early responses ously demonstrated that partial neutralization of TRAIL activity in (starting at day 7) to Pneumocystis lung infection. Furthermore, IFrag2/2 mice ameliorates the progression of BMF by seemingly we found IL-12, IL-18, and IL1b cytokine levels uniquely ele- prolonging neutrophil survival time with no effects on the pro- vated in BAL fluid from IFrag2/2 mice. However, we were re- gressive loss of progenitor cell activity (62). Consistent with this peatedly unable to detect IFN-g in BAL samples of these mice. observation, we show in this study that anti–IFN-g treatment This was also the case when time points were chosen as early as of lymphocyte-competent IFNAR2/2 mice protected from tran- days 3 and 5 postinfection (data not shown). This suggested that sient BM depression by preventing neutrophil loss and concomi- the source of early serum IFN-g was either not the lung itself or tant upregulation of TRAIL and TRAIL receptor (DR5). This was was systemically released from the lung by polarized secretion also associated with reduced extramedullary hematopoiesis. from an interstitially located cellular source (101). In this regard, In lymphocyte-deficient IFrag2/2 mice, however, the same treat- intracellular cytokine staining of phorbol ester–stimulated lung ment only ameliorated the progression of complete BMF by cells isolated from both IFrag2/2 and RAG2/2 mice over the seemingly prolonging neutrophils survival times, but with no course of infection demonstrated presence of an IFN-g–producing effects on the concomitant loss of progenitor activity. These data NK-like innate cell subset coexpressing CD49b/CD11c/CD11b support our two-hit hypothesis in the pathogenesis of BMF in our and MHC II in both animal groups (data not shown). Interest- model: the first hit includes a deregulated IFN-g systemic re- ingly, NK cells have recently been shown to provide immunity to sponse, possibly communicating the upregulation of TRAIL in the Pneumocystis lung infection (102), and type I IFNs contribute to BM. The second hit however, appears to affect early hematopoi- NK cell activation and their homeostatic control (103). Thus, etic progenitor cell activity and their ability to replenish lost cells. given a cellular composition with similar potential for IFN-g se- This seems to be specifically protected by B cell–mediated cretion within the lung of IFrag2/2 and RAG2/2 mice, it is pos- mechanisms in lymphocyte-competent IFNAR2/2 mice and may sible that these cells may be differentially regulated depending on be facilitated via protection of the HSC niche (61, 62). whether innate immune responses to Pneumocystis are elicited in A deregulated systemic IFN-g response appears to be only one the absence or presence of type I IFN signaling. This is currently contributing aspect mediating the progression of BMF in IFrag2/2 under further investigation. mice (first hit). However, by understanding upstream events of its IL-12, IL-18, and IL-1b are produced by innate immune cells deregulation we thought to gain insights into early innate pul- such as macrophages in response to activation by pattern-recognition 10 INFLAMMASOME-MEDIATED IMMUNE RESPONSES TO PNEUMOCYSTIS receptors such as TLRs (72, 75, 104). However, whereas IL-12 can apoptosis in Fanconi anemia type C-deficient cells is dependent on apoptosis signal-regulating kinase 1. Blood 106: 4124–4130. be immediately secreted in its active form, IL-18 and IL-1b re- 5. Du, W., Z. Adam, R. Rani, X. Zhang, and Q. Pang. 2008. Oxidative stress in quire the cleavage of their precursor to their active form via Fanconi anemia hematopoiesis and disease progression. Antioxid. Redox Sig- inflammasome-induced caspase-1 activation (75). Interestingly, nal. 10: 1909–1921. 6. Zheng, P., X. Chang, Q. Lu, and Y. Liu. 2013. 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