Mycobacterium tuberculosis PPE18 Protein Reduces Inflammation and Increases Survival in Animal Model of Sepsis

This information is current as Asma Ahmed, Komal Dolasia and Sangita Mukhopadhyay of October 1, 2021. J Immunol published online 18 April 2018 http://www.jimmunol.org/content/early/2018/04/17/jimmun ol.1602065 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/04/17/jimmunol.160206 Material 5.DCSupplemental

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 18, 2018, doi:10.4049/jimmunol.1602065 The Journal of Immunology

Mycobacterium tuberculosis PPE18 Protein Reduces Inflammation and Increases Survival in Animal Model of Sepsis

Asma Ahmed,*,1 Komal Dolasia,*,†,1 and Sangita Mukhopadhyay*

Mycobacterium tuberculosis PPE18 is a member of the PPE family. Previous studies have shown that recombinant PPE18 (rPPE18) protein binds to TLR2 and triggers a signaling cascade which reduces levels of TNF-a and IL-12, and increases IL-10 in macrophages. Because TNF-a is a major mediator of the pathophysiology of sepsis and blocking inflammation is a possible line of therapy in such circumstances, we tested the efficacy of rPPE18 in reducing symptoms of sepsis in a mouse model of Escherichia coli–induced septic peritonitis. rPPE18 significantly decreased levels of serum TNF-a, IL-1b, IL-6, and IL-12 and reduced organ damage in mice injected i.p. with high doses of E. coli. Peritoneal cells isolated from rPPE18-treated mice had

characteristics of M2 macrophages which are protective in excessive inflammation. Additionally, rPPE18 inhibited disseminated Downloaded from intravascular coagulation, which can cause organ damage resulting in death. rPPE18 was able to reduce sepsis-induced mortality when given prophylactically or therapeutically. Additionally, in a mouse model of cecal ligation and puncture–induced sepsis, rPPE18 reduced TNF-a, alanine transaminase, and creatinine, attenuated organ damage, prevented depletion of monocytes and lymphocytes, and improved survival. Our studies show that rPPE18 has potent anti-inflammatory properties and can serve as a novel therapeutic to control sepsis. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/ epsis is a syndrome caused by an abnormal host response to nitrogen radical intermediates, components of the complement infection. The infection is mostly due to bacteria but can cascade, catecholamines, histamines, and others. The chemical S also be due to viruses, fungi, or parasites. It is a major cause mediators can cause local damage to cells and systemic toxic of mortality especially in hospitals (1). Many survivors have effects (3–5). Although sepsis is known to be a complex condition profound disabilities, such as amputated limbs, blindness, and which involves immune suppression contributing to morbidity and cognitive problems. Sepsis leads to an overwhelming inflamma- mortality (6), the excessive production of proinflammatory cyto- tory response in the host, which manifests as well-known clinical kines, the primary being TNF-a and IL-1, are responsible for symptoms (fever, tachycardia, leukocytosis) and the accompany- the pathophysiology of sepsis, which includes vasodilation and by guest on October 1, 2021 ing systemic inflammatory response syndrome (2). In sepsis, in- increased capillary permeability further leading to hypotension, fection overstimulates the host immune response by activating hemoconcentration, macromolecular extravasation, cardiac dys- monocytes/macrophages, neutrophils, and endothelial cells. The function, and multiple organ failure. Also, inflammation-induced activation of these cells results in an elaborate and extensive ar- dysregulation of the coagulation system can result in disseminated ray of proinflammatory mediators, which includes cytokines such intravascular coagulation (DIC) (7). Injection of TNF-a and IL-1 as TNF-a, IL-1b, IL-6, and IL-8, as well as lipids, oxygen and in experimental animals leads to development of a shock-like state characterized by pulmonary edema and hemorrhage (8). Similarly *Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diag- in humans, administration of TNF-a leads to systemic inflam- nostics, Hyderabad - 500039, Telangana State, India; and †Graduate Studies, Manipal matory response syndrome (9, 10). Clinical trials in sepsis patients Academy of Higher Education, Manipal - 576104, Karnataka, India have shown benefits of anti–TNF-a Ab (11). In patients with se- 1A.A. and K.D. contributed equally to this work. vere sepsis, anti–TNF-a Ab reduced mortality and in patients Received for publication December 7, 2016. Accepted for publication March 14, suffering from shock, there was a trend for better survival with use 2018. of anti–TNF-a Ab (12). Also, reduction in levels of TNF-a and This work was supported by the Department of Science and Technology, Government overall inflammation correlates with increased survival in animal of India (Grant SB/SO/HS/0120/2010), the Department of Biotechnology, Govern- ment of India (Grant BT/PR11605/NNT/28/758/2014), and a core grant from the models of sepsis (13–15). Indeed, an exaggerated and dysregu- Centre for DNA Fingerprinting and Diagnostics by the Department of Biotechnology lated inflammatory response poses a challenge in sepsis and efforts (to S.M.); by a Research Associate fellowship from the Department of Biotechnol- need to be geared toward identifying effective anti-inflammatory ogy, Government of India (to A.A.); and by a fellowship from the Indian Council of Medical Research, Government of India (to K.D.). agents which can reduce TNF-a and inflammation for successful Address correspondence and reprint requests to Dr. Sangita Mukhopadhyay, resolution of sepsis (16–20). Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics, Inner We have earlier demonstrated that a Mycobacterium tubercu- Ring Road, Uppal, Hyderabad - 500039, Telangana State, India. E-mail address: losis protein belonging to the PPE family, PPE18, also known as [email protected] Mtb39a (Rv1196), binds to TLR2 and causes IL-10 induction in The online version of this article contains supplemental material. macrophages via activation of p38 MAPK (21). Also, its inter- Abbreviations used in this article: ALT, alanine transaminase; aPTT, activated partial thromboplastin time; CLP, cecal ligation and puncture; DIC, disseminated intravas- action with TLR2 leads to phosphorylation of suppressor of cy- cular coagulation; FBG, fibrinogen; NOS2, NO synthase 2; PLT, platelet; PT, pro- tokine signaling 3 (SOCS3), which then physically interacts with thrombin time; rPPE18, recombinant PPE18; SOCS3, suppressor of cytokine the IkBa–NF-kB/rel complex, thus preventing phosphorylation signaling 3. and degradation of IkBa and nuclear translocation of p50 and p65 Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 NF-kB and c-rel transcription factors. As a consequence of this,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602065 2 rPPE18 ATTENUATES SEPSIS there is downregulation of transcription of NF-kB–regulated genes centrifugation first at 2500 rpm to remove mouse peritoneal cells and next such as IL-12 and TNF-a (22). PPE18 selectively downregulates at 5000 rpm to remove E. coli bacteria. Peritoneal lavage cells were used proinflammatory immune responses. At the same time, it increases for Western blot analysis and cell free clear lavage was used for ELISA. secretion of IL-10 (21), which is an anti-inflammatory cytokine. CLP for inducing polymicrobial sepsis Also, our earlier work demonstrated that macrophages infected 2/2 CLP was performed as described earlier (26). Briefly, 8–12-wk-old male, with ppe18 M. tuberculosis produce less IL-10 and more IL-12 weight-matched BALB/c mice were anesthetized by administration of ket- p40 compared with those infected with wild-type M. tuberculosis amine HCl (Troika Pharmaceuticals, Dehradun, India) and xylazine HCl (21). In M. tuberculosis infection, anti-inflammatory proteins such (Indian Immunologicals, Hyderabad, India). A midline abdominal incision as PPE18 likely reduce levels of host protective proinflammatory was made under sterile conditions and the cecum below the ileocecal valve was ligated with suture, thereby preserving intestinal continuity. Once li- cytokines such as TNF-a, thus aiding survival of the bacilli (23). gated, the cecum was punctured through with a 16 G needle twice. The These same properties of PPE18 can be exploited to dampen the cecum was repositioned, the peritoneum was closed with absorbable effects of extreme inflammation observed in situations such as suture, and the skin was closed using silk suture. Sham-operated mice sepsis. With this rationale, we decided to test recombinant PPE18 underwent the same procedure including opening of the peritoneum and exposing the bowel but without ligation and needle perforation of the (rPPE18) as a therapeutic in a mouse model of sepsis. Our studies cecum. One milliliter sterile saline was injected s.c. immediately after revealed that treatment of mice with rPPE18 reduces TNF-a surgery. rPPE18 (100 mg per mice) or an equivalent volume of PBS was levels, generates M2 macrophages, inhibits DIC, and improves injected i.p. at specific times after CLP procedure. Survival rates were clinical symptoms and survival of mice suffering from septicemia determined over a period of 16 d with assessment every 12 h. From an induced by i.p. injection of a high dose of Escherichia coli. Ad- independent set of mice, blood was collected 16 h post-CLP procedure for measurement of serum ALT, creatinine, and complete blood count ditionally, rPPE18 reduced TNF-a, alanine transaminase (ALT),

analysis. Peritoneal lavage was collected in 1.5 ml sterile saline 16 h Downloaded from creatinine, and organ damage and improved survival in a mouse post-CLP procedure for measuring TNF-a. model of cecal ligation and puncture (CLP)-induced poly- ELISA for IL-1b, IL-6, IL-10, IL-12 p70, and TNF-a microbial sepsis. In light of these observations, we believe that rPPE18 or peptides derived from it may have the potential for Cytokines were quantified in sera, peritoneal lavage, and cell culture su- being developed as antisepsis therapeutics. pernatants by sandwich ELISA according to the manufacturer’s instructions as described earlier (21, 22). Absolute concentrations of TNF-a (eBio- science, San Diego, CA), IL-1b (BD Biosciences, Sparks, MD), IL-6 http://www.jimmunol.org/ Materials and Methods (PeproTech, Rocky Hill, NJ), IL-12 p70 (eBioscience), and IL-10 (BD Purification of rPPE18 Biosciences) cytokines were measured using a standard curve provided by the manufacturers. The rPPE18 was purified as described earlier (21). In brief, 6X-histidine–tagged PPE18 cloned in pRSETa was overexpressed in E. coli BL21 (DE3) cells. Histopathology Protein expression was induced by addition of 1 mM isopropyl b-D-1- thiogalactopyranoside (Sigma-Aldrich, St. Louis, MO) to log phase cul- Liver and spleen were aseptically removed from euthanized mice, fixed in tures, which were grown further for 3 h. Bacterial cells were pelleted 10% neutral buffered formalin and then embedded in paraffin wax. Sections down, resuspended in PBS (pH 7.4) containing 0.3 mg/ml lysozyme were then prepared and stained with H&E for visualizing cell death and (Sigma-Aldrich) and 1 mM PMSF (Sigma-Aldrich), and incubated at 37˚C organ damage that accompanies septicemia (27). Sections were visualized under a Nikon DS-Fi1 microscope (Shinagawa, Tokyo, Japan). Micro- for 20 min. Next, 1% sodium lauryl sarcosine (Sigma-Aldrich) was added by guest on October 1, 2021 and the mixture was sonicated. TALON (Clontech Laboratories, Mountain photographs were taken using a camera attached to the microscope. View, CA) beads were added to the supernatant obtained postsonication. Cell culture rPPE18-bound TALON beads were washed with 20 mM imidazole (Sigma-Aldrich) to remove bound impurities. rPPE18 was then eluted with BALB/c mice (6–8 wk old) of either sex were injected i.p. with 1 ml of 4% 200 mM imidazole. Eluted fractions were resolved on a gel and stained sterile thioglycollate broth (HiMedia Laboratories, Mumbai, Maharashtra, with Coomassie brilliant blue to assess purity. Eluted rPPE18 was exten- India). After 4 d, mice were sacrificed and peritoneal macrophages were sively dialyzed against PBS to remove imidazole. Dialyzed protein was harvested as described earlier (28). Macrophages were seeded either in 96- incubated with 10% v/v polymyxin B-agarose (Sigma-Aldrich) beads, as well plates at a density of 0.2 3 106 or in 24-well plates at a density of described earlier, to remove contaminating LPS (21). Before use, the 0.8 3 106 and were treated with different concentrations of rPPE18 for polymyxin B-agarose slurry was washed extensively with sterile PBS to 1 h and then activated with 1 mg/ml of bacterial LPS (Sigma-Aldrich). Cells remove any unconjugated polymyxin B present in the slurry. After incu- were cultured in RPMI 1640 medium (HyClone, GE Healthcare, Chicago, bation with protein, beads were pelleted down by centrifugation and LPS- IL) supplemented with 10% FBS (HyClone), HEPES (Sigma-Aldrich), free protein was carefully removed. Purified protein was filter-sterilized glutamine (Sigma-Aldrich), and antibiotic-antimycotic mixture (Sigma- using a 0.4-mm syringe filter and then used for experiments. The protein Aldrich) and maintained under sterile conditions of 37˚C, 5% CO2,and concentration was determined using a kit that employs the bicinchoninic 85% humidity. After 24 h, culture supernatants were collected for estimation acid method of protein estimation (Thermo Fisher Scientific, Waltham, of NO and cytokines, whereas cells were harvested for Western blotting. MA). In some cases, purified rPPE18 was subjected to repeated heating (100˚C) and freezing (liquid N2) to obtain denatured protein which was Coagulation tests used as control. For some experiments BSA was treated with polymyxin B-agarose as described above and used as control. Blood samples were collected retro-orbitally from mice in either 4% EDTA or 3.2% sodium citrate to collect whole blood and plasma, respectively. E. coli–induced septic peritonitis Blood in sodium citrate was immediately centrifuged for plasma collection to measure prothrombin time (PT), fibrinogen (FBG), and activated partial BALB/c mice were maintained at the animal house facility of Vimta Labs, thromboplastin time (aPTT) by Sysmex CA-50 Automated Blood Coag- Hyderabad and the experimental protocols were approved by and performed ulation Analyzer (Sysmex, Kobe, Japan). From whole blood, WBC and as per the guidelines of the Institutional Animal Ethics Committee of Vimta platelet (PLT) counts were determined by Medonic CA-620 hematology Labs, Hyderabad. Log phase E. coli BL21 cells were washed, resuspended analyzer (Medicon Ireland, Newry, Northern Ireland, U.K.). in 0.1 ml of sterile PBS, and injected i.p. in age- and weight-matched male BALB/c mice (8–12 wk old) at a dose of either 2.5 3 108 or 5.0 3 108 Differential leukocyte counts in CLP mice CFU to induce septic peritonitis as described by others (24, 25). Mice were administered native or denatured rPPE18 (100 mg per mice) or an equiv- For differential leukocyte count, blood was collected in EDTA tubes and alent volume of PBS (as buffer control) 1 h before or after injection with analyzed on automatic cell analyzers, ADVIA 2120 (Hematology System; E. coli. Mice were bled retro-orbitally at specific times. Blood was left to Siemens Healthcare Diagnostics, Forchheim, Germany). coagulate at room temperature for ∼2 h. Sera were then separated by Western blotting centrifugation and stored at 220˚C until further use in ELISA. For some experiments, the peritoneal cavity of euthanized mice was washed with For Western blotting, cells were lysed using cold lysis buffer comprising PBS, 5 ml sterile PBS to collect lavage. Clear lavage was obtained by sequential 1% NP-40, and protease inhibitor mixture (Roche, Penzberg, Germany). The Journal of Immunology 3

Cells were kept in lysis buffer on ice for 10 min and then subjected to one Results round of freeze-thaw after which the lysates were centrifuged at 12,000 rpm Mice pretreated with rPPE18 have reduced proinflammatory for 10 min to remove debris. Approximately 30–40 mg of proteins were separated on a 12% SDS-PAGE and then transferred on to a nitrocellulose cytokines, lesser degree of organ damage, and better clinical membrane. After blocking overnight with 5% fat-free milk, membranes were features after induction of septicemia probed with anti–NO synthase 2 (NOS2) Ab or anti–arginase-1 Ab or Earlier work had demonstrated that rPPE18 reduces LPS-induced anti–b-actin Ab (all from Santa Cruz Biotechnology, Santa Cruz, CA) fol- TNF-a levels in human macrophages (22). Similar to human lowed by an appropriate HRP-conjugated secondary Ab (Sigma-Aldrich) and then developed using ECL reagent (GE Healthcare). For detecting total and macrophages, rPPE18 inhibited TNF-a induction in mouse mac- phosphorylated p38 MAPK levels, lysates were prepared in a lysis buffer rophages activated with LPS in vitro in a dose-dependent manner containing 20 mM HEPES (Sigma-Aldrich), 250 mM NaCl (Sigma- (Supplemental Fig. 1A). Also, the suppression in TNF-a was Aldrich), 1% NP-40 (Sigma-Aldrich), 2 mM EDTA (Sigma-Aldrich), observed at both early (3 h) and late (24 h) time points post- aprotinin (Sigma-Aldrich), leupeptin (Sigma-Aldrich), DTT (Sigma- activaton with LPS (Supplemental Fig. 1B). The effects were Aldrich), and PMSF and sodium orthovanadate (both Sigma-Aldrich). found to be specific to rPPE18 as polymyxin B–treated BSA failed After resolving on a 12% SDS-PAGE, proteins were transferred onto a nitrocellulose membrane and blocked with 5% BSA (Sigma-Aldrich). The to do so (Supplemental Fig. 1C). Next, we studied if rPPE18 by membranes were then incubated with Abs to either phospho-p38 MAPK virtue of its ability to reduce TNF-a could be efficacious in re- (BD Pharmingen, San Jose, CA) or total p38 MAPK (BD Pharmingen). ducing severity of sepsis. To test this hypothesis, we employed an Membranes were washed and incubated with HRP-conjugated anti-mouse animal model of peritonitis, where mice were injected i.p. with Ab (Sigma-Aldrich) and developed using ECL reagent (GE Healthcare). high doses of Gram-negative E. coli BL21 bacteria (30). There- Arginase assay fore, we next studied the effects of rPPE18 on E. coli BL21– induced sepsis. Peritoneal administration of high numbers of Downloaded from Arginase activity in cell lysates was measured using a commercial kit 3 6 E. coli BL21 is known to result in systemic inflammation (24, 25) (QuantiChrom Arginase Assay Kit; Bioassay Systems, CA). Briefly, 0.8 10 8 thioglycollate-elicited peritoneal macrophages were treated with 3 mg/ml and we also found that i.p. injection of 2.5 3 10 E. coli BL21 rPPE18, 1 h prior to being activated with 1 mg/ml LPS. After 24 h, cells CFU resulted in a rapid rise in the serum TNF-a levels by 3 h were detached from the wells, washed, and lysed in a buffer containing (Fig. 1A) and dipped by 24 h (Fig. 1A). However, at both the early Tris-HCl (pH 7.4) (Sigma-Aldrich), 0.4% Triton X-100 (Sigma-Aldrich), and late time points tested, mice treated with rPPE18 before in- and protease inhibitor mixture (Roche). Lysates were centrifuged for 10 min at 12,000 rpm at 4˚C. The resultant supernatant was used for the duction of septicemia showed significant reduction in TNF-a http://www.jimmunol.org/ arginase assay after protein estimation using bicinchoninic acid reagent (Fig. 1A). Similar reduction in TNF-a levels upon rPPE18 treat- (Thermo Fisher Scientific). The assay was performed according to the ment was also observed in peritoneal lavage (Fig. 1B). Use of manufacturer’s instructions using 10 mg of protein. The assay measures denatured rPPE18 could not significantly reduce TNF-a levels in urea generated as a result of arginase activity as a chromogenic product whose absorbance is measured at 430 nm. Cell lysates were incubated with septic mice confirming that the observed effect was indeed due to substrate in the presence of manganese and urea generation was allowed signals triggered by rPPE18 and not by some contaminant in the for 2 h at 37˚C. The urea formed was converted to a chromogenic product protein preparation (Fig. 1A, 1B). IL-1b levels were also mea- by addition of urea stop reagent and incubated for a further 1 h at room sured in infected mice either left untreated or treated with rPPE18. temperature. Arginase activity was calculated with respect to a urea standard and expressed as U/L. IL-1b is a proinflammatory cytokine whose levels rise during peritonitis and LPS-induced septicemia (24, 31, 32). Predictably, by guest on October 1, 2021 ALT and creatinine measurement peritoneal infection with E. coli BL21 resulted in elevated serum Levels of ALT were measured in sera of septic mice subjected to E. coli levels of IL-1b which were reduced upon pretreatment with BL21–induced septic shock using a commercial ALT assay kit according rPPE18 in both serum and peritoneal lavage (Supplemental Fig. to the manufacturer’s instructions (Sigma-Aldrich). ALT activity was 2A, 2B). Another cytokine that is routinely studied as a marker for measured as a coupled enzymatic reaction, which results in product for- inflammatory responses is IL-6, levels of which are elevated during mation measured colorimetrically. ALT enzyme activity was proportional to pyruvate generated. Concentration of pyruvate generated was calculated sepsis (33). Treatment with rPPE18 before induction of sepsis with the help of a pyruvate standard curve. In brief, BALB/c mice were resulted in decreased IL-6 induction in sera and peritoneal lavage injected i.p. with 100 mg of rPPE18 or an equivalent volume of PBS before (Supplemental Fig. 2C, 2D). Pretreatment with rPPE18 was able to 3 8 3 8 being administered either 2.5 10 or 5.0 10 E. coli BL21 CFU. After inhibit the proinflammatory cytokine storm not only at 2.5 3 108 3 or 24 h, mice were bled retro-orbitally and sera were separated and used 3 8 for ALT assay. The assay was performed for 45 min at 37˚C. ALT enzyme CFU but also at a higher dose of 5.0 10 E. coli BL21 CFU activity was expressed as U/L. For experiments involving CLP, analysis for (Supplemental Fig. 2E–G) as observed by reduction in serum levels creatinine and ALT in sera was done on Johnson & Johnson Vitros 250 of IL-1b (Supplemental Fig. 2E), IL-6 (Supplemental Fig. 2F), and Chemistry Analyzer. TNF-a (Supplemental Fig. 2G) at 3 h postinfection. Clinical features of septic mice and survival studies in To assess the effect of rPPE18 on sepsis-induced organ damage, E. coli–induced sepsis levels of serum ALT, a marker for liver damage, were determined (18, 19, 32). As expected, injection of 2.5 3 108 E. coli BL21 BALB/c mice were injected with rPPE18 (100 mg) or PBS and 1 h later infected with 2.5 3 108 or 5.0 3 108 E. coli BL21 CFU. After 24 h, resulted in exacerbated liver injury as indicated by elevated serum clinical symptoms, such as conjunctivitis, ruffling of fur coats, and activity ALT activity at 24 h postinfection. However, mice which had re- on stimulation, were observed in a group of 10 mice and graded as de- ceived rPPE18 prior to i.p. injection of E. coli BL21 had signifi- scribed earlier (29). A collective grade was assigned to each group cantly reduced levels of serum ALT indicating protection against depending on the severity of the symptom observed in the majority of the animals. To evaluate the effect of rPPE18 on survival of mice, E. coli sepsis-induced liver damage (Fig. 1C). The severity of liver BL21–infected mice were administered rPPE18 1 h pre- or postinfection. damage as reflected in elevated serum ALT levels was also ob- Survival of mice was monitored every 6–8 h. Time of death was noted as served in histological sections of tissue obtained from septic mice. accurately as possible. The H&E-stained sections of the liver (Fig. 1D) and spleen 8 Statistical analysis (Fig. 1E) from mice infected i.p. with 2.5 3 10 E. coli BL21 CFU showed severe lesions and damage resulting from severe sepsis. In The nonparametric Mann–Whitney two-tailed test was performed to de- contrast, mice which had received rPPE18 prior to injection with termine significance. The log rank (Mantel–Cox) test was performed to 8 determine significant differences in survival curves. The p values ,0.05 2.5 3 10 E. coli BL21 CFU had almost normal tissue architecture were considered significant. (Fig. 1D, 1E). Even at a high dose of 5.0 3 108 E. coli BL21 4 rPPE18 ATTENUATES SEPSIS Downloaded from http://www.jimmunol.org/

FIGURE 1. rPPE18 inhibits TNF-a and organ damage in vivo in septic mice. BALB/c mice were given either 100 mg of native rPPE18 or denatured rPPE18 (D-rPPE18) or an equivalent volume of PBS i.p. prior to infection with 2.5 3 108 E. coli BL21 CFU via the i.p. route. The uninfected control group received equivalent volume of PBS alone. TNF-a levels in sera were measured by ELISA at 3 and 24 h postinfection (A). At 24 h postinfection, TNF-a levels were measured in peritoneal lavage by ELISA (B). At 24 h postinfection, mice were bled retro-orbitally and sera were separated to measure ALT levels (C). Also, after 24 h mice were sacrificed and liver (D) and spleen (E) sections were prepared and stained with H&E. Photographs of representative sections visualized at 1003 magnification are shown. Data shown in (A)–(C) are mean 6 SEM from multiple mice. by guest on October 1, 2021 CFU–induced sepsis, rPPE18 protein was able to inhibit ALT of M1 macrophages that are inflammatory and produce cytokines levels in sera (Supplemental Fig. 3A), as well as spleen and liver such as TNF-a, IL-1b, IL-6, and IL-12. On the other hand, cyto- organ damage as assessed by histopathological analysis of tissue kines such as IL-4, IL-10, and IL-13 activate macrophages through sections (Supplemental Fig. 3B, 3C). the alternate pathway giving rise to M2 macrophages (36, 37). Severity of liver and spleen damage ascertained by measurement Elevated numbers of M1 macrophages and high levels of M1- of ALT activity and observation of H&E-stained tissue sections derived cytokines are correlates of sepsis severity (38, 39). On was found to correlate with external clinical features of septic the other hand, M2 macrophages which produce anti-inflammatory mice. Mice suffering from sepsis exhibit clinical features which mediators and promote tissue regeneration (40) are crucial for are reflective of the shock that they are experiencing. The clinical resolution of sepsis (38, 39). M1 macrophages have high NOS2 features such as conjunctivitis, ruffling of hair coat, huddling, and levels, whereas M2 macrophages have high arginase-1 levels (41). activity upon stimulation can be graded to reflect the severity of Because rPPE18 inhibits production of proinflammatory cytokines sepsis (29). Groups of 10 mice were examined at 24 h postin- such as TNF-a, IL-1b, and IL-6 (Fig. 1, Supplemental Fig. 2), we jection of either 2.5 3 108 or 5.0 3 108 E. coli BL21 CFU. next explored the possibility of rPPE18 promoting the polarization Comparisons were made between groups which either received of macrophages toward the M2 phenotype. To determine the effect PBS or rPPE18 prior to administration of E. coli BL21. A grade of of rPPE18 on polarization of macrophages, initially levels of normal, mild, marked, and severe was given depending on the arginase-1 and NOS2 in mouse macrophages activated (in vitro) severity of the clinical symptoms. The observations (Supplemental with LPS in the absence or presence of rPPE18 were compared. Fig. 2H) and grades are summarized in Table I, which clearly Indeed, NOS2 and arginase-1 were found to be reciprocally regu- indicate that mice pretreated with rPPE18 are clinically healthier lated in macrophages treated with rPPE18 (Fig. 2A). In LPS- with reduced sepsis symptoms. However, rPPE18 by itself had no activated macrophages, rPPE18 increased arginase-1 and decreased effect on the kinetics of bacterial growth in vitro (Supplemental NOS2 in a dose-dependent manner (Fig. 2A). Also, decreased Fig. 2I) indicating that the observed ameliorative effect of rPPE18 NOS2 levels correlated with reduced NO production in LPS- is due to its ability to modulate the host immune responses. activated macrophages treated with rPPE18 when compared with macrophages activated with LPS alone (Fig. 2B). As compared with rPPE18 triggers alternate activation of macrophages LPS-treated macrophages, arginase activity was higher in rPPE18 Macrophages are innate cells that are exposed to various microbes plus LPS–treated macrophages (Fig. 2C) which correlated with and microbial products (34). The varying stimuli that act on increased arginase-1 protein levels in these cells (Fig. 2A). macrophages can trigger different activation programs leading to Previous work in our laboratory demonstrated that rPPE18 after generation of subsets of macrophages with specific phenotypes and binding to TLR2 increased phosphorylation of p38 MAPK, which functions (35). LPS and IFN-g stimulation results in generation in turn was responsible for downstream activation of IL-10 (21). The Journal of Immunology 5

Table I. Mice treated with rPPE18 appear clinically healthier after induction of septic shock

Parameter PBS rPPE18 Conjunctivitis 5.0 3 108 (severe) 5.0 3 108 (marked) Extreme (Both eyes closed with heavy serous discharge) Marked (Both eyes open with serous discharge) 2.5 3 108 (marked) 2.5 3 108 (mild) Marked (Both eyes open with serous discharge) Mild (Usually serous discharge present in one eye, eyes were open) Hair coat 5.0 3 108 (severe) 5.0 3 108 (marked) Extremely ruffled coat, ruffling evident as early as 3 h. Moderately ruffled coats at 20 h. No ruffling seen at 3 h. 2.5 3 108 (marked) 2.5 3 108 (mild) Marked ruffling observed at 20 h. Mild ruffling in majority of animals. Some animals had shiny unruffled coats. Activity upon stimulation 5.0 3 108 (severe) 5.0 3 108 (marked) Moribund, extremely sluggish, animals exhibited Sluggish activity. Animals exhibited huddling behavior huddling behavior, did not respond or move to but upon stimulation responded and broke out of huddle. stimulation. 2.5 3 108 (marked) 2.5 3 108 (Normal) Sluggish, animals exhibited huddling behavior. Animals remained normally active. However, upon stimulation animals responded by raising their heads and breaking out of the huddle.

8 8 BALB/c mice were given either PBS or 100 mg of rPPE18 i.p. prior to infection with either 2.5 3 10 or 5.0 3 10 E. coli BL21 CFU. Twenty-four hours later parameters Downloaded from listed in the table were observed. Each group consisted of five mice. Mice were graded according to the severity of the symptom: normal, mild, marked, and severe.

The phosphorylation of p38 MAPK was reconfirmed in our system harvested from E. coli–treated but rPPE18-untreated mice of study (Fig. 2D). Addition of rPPE18 to thioglycollate-elicited (Fig. 3C). It has been reported that LPS stimulation and sepsis mouse peritoneal macrophages resulted in increased p38 MAPK induces arginase-1 expression (45). However, there are also re- http://www.jimmunol.org/ phosphorylation (Fig. 2D, lane 3). Addition of LPS also led to ports which show endotoxin-mediated arginase-1 suppression in increased p38 MAPK phosphorylation (Fig. 2D, lane 2). However, macrophages (46). It is evident in our system of study that in in the presence of rPPE18, phospho-p38 MAPK levels were peritoneal exudate cells from E. coli–treated and rPPE18- greater in LPS-activated cells (Fig. 2D, lane 4). Next, we exam- untreated mice, there is elevated NOS2 which could be the pos- ined whether activation of p38 MAPK by rPPE18 was crucial for sible reason of repressed arginase-1 expression. Overall, these the elevated arginase-1 levels in these cells. For this purpose, results suggest that rPPE18 leads to generation of IL-10hiNOS2lo SB203580, a specific p38 MAPK inhibitor, was used. The de- arginase-1hi M2 macrophages in mice suffering from septicemia. crease in arginase-1 with SB203580 in cells treated with just LPS by guest on October 1, 2021 was not very evident (Fig. 2E, lanes 5 and 6). However, pre- Pretreatment with rPPE18 decreases DIC and increases treatment with SB203580 resulted in reduction of arginase-1 survival in septic mice levels in cells administered both rPPE18 and LPS (Fig. 2E, In sepsis, the proinflammatory cytokine surge is known to cause lanes 8 and 10). These results suggest that the rPPE18-mediated activation of coagulation leading to DIC (47). Development of DIC increase in arginase-1 level is due to downstream signaling events and its severity correlates with mortality in severe sepsis (48, 49). triggered by phosphorylation of p38 MAPK. DIC is characterized by systemic activation of blood coagulation, So far, it was observed that rPPE18 decreased NOS2 expression which results in generation and deposition of fibrin, leading to and increased arginase-1 expression and its activity in LPS- microvascular thrombi in various organs, depleting the organ’s stimulated mouse peritoneal macrophages (Fig. 2A–C) indicat- supply of essential nutrients and oxygen, thus contributing to ing that rPPE18 could cause generation of M2 macrophages multiple organ dysfunction syndrome (50). Due to extensive co- in vitro. Apart from increased arginase-1 levels, M2 macrophages agulation, finally a stage arrives where consumption and subse- secrete higher amounts of IL-10 (42), whereas M1 macro- quent exhaustion of coagulation proteins and PLTs (from ongoing phages secrete higher amounts of IL-12 (35, 43, 44). In the next activation of coagulation) may lead to severe bleeding (48). experiment, therefore, effect of rPPE18 on macrophage polariza- Studies have shown that IL-6 can cause activation of coagulation tion in the in vivo system of sepsis was tested. IL-10 and IL-12 (51, 52), whereas TNF-a and IL-1b inhibit the anticoagulation p70 levels were measured in sera of mice which either received cascades (53, 54). Because rPPE18 inhibits IL-6, TNF-a, and PBS or rPPE18 before being injected with 2.5 3 108 or 5.0 3 108 IL-1b, responsible for disturbance of coagulation and anti- E. coli BL21 CFU. IL-10 levels were higher (Fig. 3A), whereas coagulation signaling events, the effect of rPPE18 on DIC during IL-12 p70 levels were lower (Fig. 3B) in sera of septic mice which sepsis was examined. For this, BALB/c mice were administered had received rPPE18. Next, levels of NOS2 and arginase-1 in the rPPE18 1 h before infection with 2.5 3 108 E. coli BL21 CFU and peritoneal exudate cells harvested from these mice were measured. after 12 h, blood was collected to evaluate coagulation parameters Mice that received a combination of PBS and E. coli BL21 for DIC such as PT, aPTT, FBG, PLT count, and WBC count. showed higher NOS2 levels compared with mice which received Prophylactic administration of rPPE18 helped reduce clotting time rPPE18 and E. coli BL21 (Fig. 3C, upper panel). Conversely, as evident by decreased PT (Fig. 4A) and aPTT (Fig. 4B) values in arginase-1 levels were higher in cells from mice which received a the rPPE18-treated group as compared with PBS-treated group. combination of rPPE18 and E. coli BL21 CFU compared with Moreover, in mice infected with E. coli, rPPE18 increased FBG cells from mice which received PBS and E. coli BL21 CFU levels (Fig. 4C), PLT count (Fig. 4D), and WBC count (Fig. 4E). (Fig. 3C, middle panel). Even though arginase-1 expression was Therefore, rPPE18 inhibits DIC in septic mice. evident in peritoneal macrophages harvested 4 d post-thioglycollate Experiments carried out so far showed that pretreatment administration (Fig. 2A, 2E), it was not detected in peritoneal cells with rPPE18 could reduce severity of E. coli BL21–induced 6 rPPE18 ATTENUATES SEPSIS Downloaded from http://www.jimmunol.org/

FIGURE 2. rPPE18 treatment reduces NOS2 and increases arginase-1 via p38 MAPK. Thioglycollate-elicited macrophages were pretreated with dif- ferent concentrations of rPPE18 for 1 h before being activated with 1 mg/ml LPS. After 24 h cells were lysed and NOS2, arginase-1, and b-actin levels were observed by Western blotting. A representative blot from four independent experiments is shown (A). Additionally, NO levels in supernatants (B) and arginase-1 activity in lysates (C) were measured. Data shown are mean 6 SEM of three independent experiments. Thioglycollate-elicited macrophages by guest on October 1, 2021 were pretreated with 3 mg/ml rPPE18 for 1 h before being activated with 1 mg/ml LPS for another 1 h, after which cells were lysed and levels of phosphorylated and total p38 MAPK were studied by Western blotting (D). Thioglycollate-elicited macrophages were treated with 10 mg/ml SB203580 for 30 min followed by 1 or 3 mg/ml rPPE18 for 1 h. Next, cells were activated with 1 mg/ml LPS for 24 h, lysed, and arginase-1 levels were studied by Western blotting; b-actin was used as loading control (E). Data shown are representative of three different experiments. sepsis in terms of reducing levels of proinflammatory cytokines (Fig. 4A–E). Mice administered rPPE18 also appeared clinically (Fig. 1A, 1B, Supplemental Fig. 2), reducing organ dam- healthier (Supplemental Fig. 2H, Table I). Whether all this age (Fig. 1C–E, Supplemental Fig. 3), and preventing DIC translated to an ultimate survival advantage in mice which

FIGURE 3. rPPE18 leads to generation of M2 macrophages in vivo. BALB/c mice were in- jected with 100 mg of either rPPE18 (n =7)oran equivalent volume of PBS (n = 7) 1 h prior to i.p. administration of either 2.5 3 108 or 5.0 3 108 E. coli CFU. The uninfected control group (n =5) received equivalent amount of PBS only. After 24 h, IL-10 (A) and IL-12 p70 (B) levels in sera were measured by ELISA. The peritoneal wash was collected and cells from the peritoneal wash were lysed and Western blotting was carried out for NOS2, arginase-1, and b-actin. Each lane represents lysate from an individual mouse (C). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Prophylactic administration of rPPE18 inhibits DIC and improves survival in septic mice. BALB/c mice were either given 100 mg of rPPE18 or an equivalent volume of PBS i.p. prior to i.p. infection with 2.5 3 108 E. coli BL21 CFU. Uninfected mice and mice administered only rPPE18 were used as control. Mice were bled retro-orbitally 12 h postinduction of peritonitis and levels of clotting (DIC) parameters PT (A), aPTT (B), and FBG (C) were measured from plasma. PLT count (D) and WBC count (E) were determined from blood. Data are shown as mean 6 SEM of six mice in each group. For survival studies, BALB/c mice were given either 100 mg of rPPE18 or an equivalent volume of PBS i.p. 1 h prior to infection with either 2.5 3 108 (F)or 5.0 3 108 (G)ofE. coli BL21 CFU via i.p. route. Survival was monitored at regular intervals untill 5 d and survival curves were plotted (F and G). Each experimental group comprised of six (F) and nine (G) mice. Significance in differences of survival percentages were assessed by a log rank test for trend. received rPPE18 remained to be investigated. Therefore, it was administration of rPPE18 eventually confers survival advantage further examined whether rPPE18-pretreated mice survived during sepsis. better upon induction of sepsis. The mean survival time of mice injected with 2.5 3 108 E. coli BL21 CFU was 35 h (Fig. 4F), rPPE18 administered therapeutically reduces levels of whereas for mice injected with 5.0 3 108 E. coli BL21 CFU, it proinflammatory cytokines, inhibits DIC, and improves was 24 h (Fig. 4G), indicating that both these doses of bacteria survival of septic mice are lethal. However, mice given rPPE18 i.p. before adminis- So far for our experiments we had used a system where rPPE18 was tration of 2.5 3 108 E. coli BL21 CFU were almost completely given to mice prophylactically, that is, before induction of septi- protected from sepsis-induced lethality. Their survival was cemia by E. coli BL21. This helped in unraveling its anti- 75% when the last death in the PBS-injected group was inflammatory properties, its ability to inhibit DIC in infected recorded at 37 h (Fig. 4F). Also, in mice which were given mice, and its ability to polarize macrophages toward the M2 rPPE18 and a higher dose of 5.0 3 108 E. coli BL21 CFU, the phenotype. However, to have potential as a tool for treatment of meansurvivaltimewas46hcomparedwith24hformice sepsis, it is important to study if rPPE18 can reduce inflammation which did not receive rPPE18 (Fig. 4G). This shows that and DIC when administered therapeutically. For this, rPPE18 was 8 rPPE18 ATTENUATES SEPSIS given 1 h postinduction of sepsis and it was found that rPPE18 could Administration of high doses of E. coli BL21 led to a rapid rise in significantly reduce levels of TNF-a (Fig. 5A), IL-1b (Fig. 5B), IL- TNF-a and resulted in 100% mortality by 40 h. This model 12 p70 (Supplemental Fig. 4A), and IL-6 (Supplemental Fig. 4B) in allowed us to study the effects of rPPE18 and dissect its mecha- mice infected with 2.5 3 108 E. coli BL21, indicating that rPPE18 is nism of action in sepsis. However, we next wished to study the a potent inhibitor of proinflammatory cytokines when administered effect of rPPE18 in polymicrobial sepsis induced by CLP, which is therapeutically. Next, it was examined whether rPPE18 could also a more physiological model of sepsis (55, 56). In this model, the inhibit DIC when injected therapeutically. BALB/c mice were ad- effects of rPPE18 were studied on TNF-a levels; WBC count; ministered rPPE18 1 h postinfection with 2.5 3 108 E. coli BL21 numbers of lymphocytes, monocytes, and neutrophils; liver and CFU and 12 h later, blood was collected and coagulation parameters kidney function; and ultimately survival. for DIC were examined. Administration of rPPE18 therapeutically Polymicrobial sepsis induced by CLP increased levels of TNF-a helped to reduce clotting time as evidenced by decreased PT (Fig. in the peritoneal lavage, which were significantly reduced in mice 5C) and aPTT (Fig. 5D) values in rPPE18-treated group as compared that received rPPE18 therapy (Fig. 6A). We, however, could not with PBS-treated septic mice. Interestingly, rPPE18 also increased measure TNF-a in blood serum. This is consistent with earlier FBG levels (Fig. 5E), PLT count (Fig. 5F), and WBC count (Fig. 5G) reports (57, 58). CLP also resulted in dramatic reduction in in mice infected with E. coli in comparison to PBS-treated septic numbers of lymphocytes (Fig. 6B), monocytes (Fig. 6C), and mice. Thus, rPPE18 is able to prevent inflammation and DIC in septic neutrophils (Fig. 6D). This reduction in numbers was reflected in mice, even when administered therapeutically. the decrease in WBCs post–CLP-induced sepsis (Fig. 6E). Ad- Because rPPE18 reduced induction of both proinflammatory ministration of rPPE18 restored lymphocyte (Fig. 6B), monocyte cytokines and DIC in mice infected with a higher dose of E. coli (Fig. 6C), and WBC (Fig. 6E) numbers comparable to sham- when administered therapeutically, we next investigated its effects operated mice. CLP also resulted in reduction in blood neutro- Downloaded from on survival of these mice. Mice which were not administered phil levels. However, neutrophil numbers were not significantly rPPE18 died by 23 h postinduction of sepsis (Fig. 5H). However, but only marginally higher in rPPE18-treated mice (Fig. 6D). therapeutic administration of rPPE18 at 1, 24, and 72 h postinjec- As CLP is known to cause liver and kidney damage (59, 60), we tion of E. coli BL21 dramatically increased survival to 71% as next assessed liver and kidney function by measuring serum ALT observed at the last time point of 384 h (Fig. 5H). Thus, rPPE18 and creatinine levels. We observed an increase in serum ALT improves survival when administered therapeutically to septic mice. (Fig. 6F) and creatinine (Fig. 6G) levels 16 h post-CLP. Thera- http://www.jimmunol.org/ peutic administration of rPPE18 significantly reduced ALT and Administration of rPPE18 improves survival in a mouse model creatinine levels. This effect on ALT levels was also reflected in of polymicrobial sepsis induced by CLP differences in liver pathology between the two groups of animals. In all our studies so far, we had used a system where i.p. in- Mice subjected to CLP but not treated with rPPE18 showed jection of high doses of E. coli BL21wasusedtoinducesepsis. marked necrosis and liver damage compared with sham-operated by guest on October 1, 2021

FIGURE 5. Therapeutic administration of rPPE18 offers protection from sepsis. BALB/c mice were infected with 2.5 3 108 E. coli BL21 CFU i.p. and 1 h later were given either 100 mg of rPPE18 or an equivalent volume of PBS i.p. Levels of TNF-a (A) and IL-1b (B) in the sera were measured by ELISA 12 h postinfection. Data are shown as mean 6 SEM of seven mice in each group. In addition, 12 h postinfection levels of clotting parameters PT (C), aPTT (D), and FBG (E) were measured from plasma. PLT count (F) and WBC count (G) were determined in whole blood. Data shown are mean 6 SEM of six mice in each group. For survival studies, BALB/c mice were infected with 2.5 3 108 E. coli BL21 CFU via the i.p. route and subsequently given either 100 mg of rPPE18 or an equivalent volume of PBS i.p. at 1, 24, and 72 h. Survival was monitored at regular intervals until 16 d (384 h) and survival curves were plotted (H). Each experimental group comprised seven mice. Significance in differences in survival percentages was assessed by a log rank test for trend. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 6. Therapeutic administration of rPPE18 reduces inflammation and organ damage and reduces mortality in sepsis induced by CLP. Poly- microbial sepsis was induced by CLP in BALB/c mice. One hour after surgery mice were given 100 mg of rPPE18 or an equivalent volume of sterile PBS i.p. Sham-operated mice were used as controls in all experiments. After 16 h, mice were sacrificed and peritoneal lavage was collected and TNF-a was measured by ELISA (A). In addition, blood was collected by retro-orbital puncture after 16 h. Whole blood was used for measuring lymphocytes (B), by guest on October 1, 2021 monocytes (C), neutrophils (D), and WBCs (E). Serum was also used for measuring ALT (F) and creatinine (G). Data shown are mean 6 SEM of five to eight mice in each group. Liver sections were prepared and stained with H&E. Photographs of representative sections visualized at 1003 magnification are shown (H). For survival studies, polymicrobial sepsis was induced by CLP. One and 20 h after surgery, mice were given 100 mg of rPPE18 or an equivalent volume of sterile PBS i.p. Survival was monitored at regular intervals until 16 d (384 h) and survival curves were plotted (I). Each experimental group comprised eight mice. Significance in differences in survival percentages was assessed by a log rank test for trend. mice, whereas rPPE18-treated CLP mice had more hypertrophy uncontrolled bacteremia resulting from situations of pneumonia, (Fig. 6H), which can be a nonadverse effect (61), and hyper- peritonitis, and surgical procedures (63, 64). Specifically, in the vacuolation, which may be a host protective response (62). These majority of cases, infection is caused by Gram-negative bacteria data suggest that rPPE18 can provide protection from poly- and LPS from the outer membrane of the bacteria overstimulates microbial sepsis–induced organ damage. the host immune response. Treatment for sepsis consists of erad- Finally, we studied the effect of rPPE18 on survival of mice ication of infection through early and aggressive treatment with subjected to CLP-induced polymicrobial sepsis. Mice that received appropriate antibacterials. However, despite advances in the de- PBS post-CLP had a median survival time of 22 h, whereas those velopment of powerful antibiotics, sepsis is still life-threatening. that received rPPE18 had significantly improved survival. In the Therefore, there remains a need for development of novel anti- rPPE18 treatment group, 75% of mice were still surviving when the sepsis therapies combining antibiotic therapy with the application last death in the PBS group was registered at ∼60 h post-CLP of a nonsteroidal anti-inflammatory regimen. (Fig. 6I). Mice were monitored until 16 d and the survival per- In this study, septicemia was induced in mice by injection of very centage in the rPPE18-treated group at this time was 70% high doses (2.5 3 108 and 5 3 108 CFU) of Gram-negative E. coli (Fig. 6I). These data indicate that rPPE18 by virtue of its ability to BL21 i.p. Such high doses of bacteria were potent inducers of reduce TNF-a levels and prevent organ damage can provide proinflammatory cytokines (TNF-a, IL-1b, and IL-6) and organ protection in polymicrobial sepsis. Importantly, results from these damage, which are hallmarks of septicemia. Also, apart from in- experiments validate previous observations made using the model ducing a cytokine storm, high doses of E. coli BL21 resulted in of E. coli BL21–induced septic shock. 100% fatality by 40 h. Therefore, this is a robust model to study excessive inflammation and septicemia as has been reported pre- Discussion viously (24, 25). In this system we tested the effect of rPPE18 Sepsis is a condition described by systemic hyperinflammation belonging to the mycobacterial PE/PPE family which comprises induced because of excessive production of proinflammatory several immunomodulatory proteins (65). We found that rPPE18 cytokines such as TNF-a, IL-1b, and IL-6. Its causes are significantly reduces levels of TNF-a, IL-1b, and IL-6. Both 10 rPPE18 ATTENUATES SEPSIS

IL-1b and IL-6 have been shown to be elevated during septicemia considerably increased during M. tuberculosis infection (80–82). By (31–33). However, the role of IL-6 in experimental sepsis models upregulating arginase-1, M. tuberculosis can counteract the bacte- is controversial as IL-6 has both anti- and proinflammatory ricidal effect of NOS2/NO, and survive better inside macrophages properties (66). Blockade of IL-6 has been shown to be beneficial (83). Therefore, activation of arginase-1 is an important virulence in sepsis as well as other inflammatory diseases (66, 67) indicating mechanism of M. tuberculosis. Upregulation of arginase-1 by a positive correlation between elevated IL-6 levels and sepsis M. tuberculosis is dependent on TLR2 and STAT-6 (80). rPPE18 is severity (68, 69). Interestingly, in rPPE18-treated mice, we ob- known to bind to TLR2 and induce IL-10 (21). We now know that served reduced coagulation time, indicated by decreased PT and rPPE18-mediated signaling indeed leads to activation of arginase- aPTT, with a simultaneous increase in FBG, PLT, and WBC levels 1 and probably increases polarization of macrophages toward the during infection with E. coli. Thus, rPPE18 may reduce the in- M2 phenotype. Although this mechanism might prove useful for flammation and thereby decrease toxicity of sepsis by inhibiting M. tuberculosis survival as observed in a mouse model of infection TNF-a, IL-1b, and IL-6 production and DIC. Pretreatment with (23), it can also be used to provide protection during E. coli– rPPE18 indeed reduced sepsis-induced organ damage as studied induced and CLP-induced septicemia. Use of rPPE18 in a model both by release of ALT and histopathology. Also, these mice were of sepsis not only helped us to identify a potential candidate for clinically healthier and most importantly, rPPE18-treated septic sepsis therapy but also provided novel insights into the mechanism mice had better survival rates. of action of a mycobacterial virulence factor. In a separate mouse model of polymicrobial sepsis induced Clinical trials have shown benefits of use of anti–TNF-a Ab (11, by CLP, rPPE18 reduced TNF-a, attenuated liver and kidney 12); however, no major benefits of activated protein C and TLR4 damage, prevented sepsis-induced depletion of monocytes and antagonist eritoran were observed in sepsis treatment (84–86). lymphocytes, and ultimately increased survival. The protective Immune adjuvants such as IL-7, IL-15, and GM-CSF have Downloaded from effects of rPPE18 were, therefore, demonstrated in two models of emerged as potential therapies for sepsis (87). Immune modulators sepsis. Ability to reduce TNF-a and attenuate organ damage in such as thymosin a 1 (88) and curcumin (89–91), anticoagulants CLP-induced sepsis correlates with increased survival as has been such as heparin (92), the PI3K-Akt pathway inhibitor wortmannin, demonstrated previously (13, 14, 70–72). rPPE18 was able to LY294002 that suppresses coagulation and inflammation (93), and consistently reduce elevated levels of TNF-a in E. coli BL21– i.v. Ig (94) have shown promise in animal studies or clinical trials. induced and CLP-induced sepsis when administered therapeuti- Also, novel targets for sepsis therapy such as regulatory receptors http://www.jimmunol.org/ cally. The former is a model where LPS is likely to play a major PD-1 and BTLA have emerged (87). rPPE18 inhibits both in- role (73, 74) and the latter is a model of polymicrobial sepsis. flammation and DIC and is hence a promising therapeutic agent rPPE18 works efficiently to reduce mortality in both model sys- for treatment of sepsis. Interestingly, the N-terminal of rPPE18 tems. Timely administration of antibiotics controls bacterial rep- spanning amino acids 1–179 is sufficient to bind to TLR2 and lication, but cannot undo the damaging effects of the systemic trigger SOCS3-mediated activation of IL-10 (21, 22). Future cytokine storm. A strategy of controlling bacterial multiplication study of the therapeutic use of rPPE18 in sepsis could focus on along with inhibition of excessive proinflammatory cytokines and the 179 aa long N-terminal or its shorter segments, which might DIC by use of agents such as rPPE18 might be more effective in be more biologically effective and easier to deliver. Individuals by guest on October 1, 2021 controlling human sepsis. latently infected with M. tuberculosis or having a history of Macrophages are versatile cells. Their microbicidal function and active tuberculosis disease or vaccinated with M. bovis bacillus their participation in the inflammatory response can have immense Calmette–Gue´rin may harbor Abs to PPE18 that might hinder its bearing on the outcome of septicemia (34). In our model of E. coli future therapeutic use. In such situations peptide fragments BL21–induced sepsis, we observed accumulation of cells with which are as effective as the full length protein may be used. reduced NOS2 and elevated arginase-1 expression in the perito- Another advantage of using rPPE18 as opposed to anti–TNF-a neal cavity in rPPE18-treated mice. These are characteristics of Ab is that the efficacy of an Ab is reliant on its ability to com- M2 macrophages (35, 41, 75). M2 macrophages are immuno- pletely neutralize TNF-a, unlike agents such as rPPE18 that modulatory and have low microbicidal activity compared with M1 inhibit the generation of TNF-a. macrophages and participate in processes of wound healing (35, Proteins from pathogens have been used as vaccines for many 44). In cases of septicemia, studies have shown that a balance of years. More recently, focus has shifted to exploiting virulence protein M1 and M2 macrophages may determine the severity of sepsis and factors from bacteria and viruses for therapy of immune-related survival of infected animals (38). Previous studies using experi- disorders, coronary syndromes, and so on (95, 96). A good case mental animal models have shown that one of the underlying in point is the potential therapeutic use of viral Serp-1 and Serp-2 mechanisms of protection from sepsis is the increased proportion proteins, which have been shown to reduce vascular inflammation of M2 macrophages (38, 45, 76–78). PPE18 is capable of acting as and T cell apoptosis in a murine model (97, 98). Results from our an immunomodulator in the recombinant form as well as when study show that rPPE18 is a potent inhibitor of proinflammatory present as part of whole M. tuberculosis (21–23). Our earlier cytokines and DIC when given both prophylactically and ther- studies (21) and results presented in this study clearly show apeutically in mouse models of E. coli BL21–induced and rPPE18 to be a highly potent inducer of IL-10, a cytokine re- CLP-induced septicemia. Also, rPPE18 was found to be a potent sponsible for polarization of macrophages toward the M2 phe- inducer of arginase-1 and IL-10, markers for M2 macrophages. notype and also a cytokine that is secreted by M2 macrophages. In A therapy which inhibits the production of inflammatory cyto- addition, rPPE18 reduces levels of endotoxin- and infection- kines by polarizing macrophages to the M2 phenotype perhaps has induced IL-12 and TNF-a, also a typical characteristic of M2 a better chance of success along with antibiotics. Thus, its ability macrophages. Earlier, we demonstrated that rPPE18 reduces to dampen harmful responses and elevate protective responses in NF-kB signaling by upregulating SOCS3 protein resulting in de- sepsis makes rPPE18 a promising candidate for antisepsis therapy. creased production of IL-12 and TNF-a (22). Incidentally, a de- ficiency in SOCS3 has been found to increase polarization of Acknowledgments macrophages toward the M1 phenotype and inflammation (79). We thank Dr. Jayant P. Hole, Suman Komjeti, Sridhar Kavela, and Sravani Apart from conditions of sepsis, arginase-1 has been found to be Edula for providing help in animal experiment work. The Journal of Immunology 11

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4 1.5 2 3 ng/ml ng/ml

ng/ml 1 α α 2 1 α TNF- TNF- 0.5 1 TNF- 0 0 0

rPPE18 3 h 24 h LPS

Supplemental Figure 1. rPPE18 reduces TNF-α level in mouse macrophage cultures in vitro. Thioglycolate elicited mouse macrophages

were pre-treated with different concentrations of rPPE18 for 1 h followed by activation with 1 µg/ml LPS. After 24 h, TNF-α levels in culture

supernatants were measured by ELISA (A). Data shown are mean ± SEM of 3 independent experiments. In another experiment, thioglycolate

elicited mouse macrophages were either left untreated or pre-treated with 3 µg/ml of rPPE18 for 1 h and then activated with 1 µg/ml of LPS.

TNF-α levels in culture supernatants were measured by ELISA at 3 h and 24 h post addition of LPS (B). Data shown are mean ± SD of triplicate

wells of a representative experiment of a total of 2 performed. Thioglycolate elicited mouse macrophages were incubated with polymyxin B

treated rPPE18 or BSA at 3 µg/ml concentration for 1 h and then stimulated with LPS. After 24 h TNF-α levels were measured in culture

supernatants by ELISA (C ). Data shown are mean ± SEM from three mice. ns - not significant. ABC D

E F G

H E. coli I 1.0 PBS PBS rPPE18 0.8 rPPE18 BSA 0.6

0.4

0.2

0.0 0 2 Time (h)

Supplemental Figure 2. rPPE18 reduces symptoms of septicaemia without being directly toxic to E. coli . A-G.

Balb/c mice were either injected with PBS alone or given 100 µg rPPE18 intraperitoneally prior to infection with 2.5 ×

108 (A-D) or 5 × 108 (E-G) E. coli BL21 CFU via intraperitoneal route. At 3 h post-infection, IL-1β (A, E), IL-6 (C, F) and TNF-α (G) levels in the sera were measured by ELISA. At 24 h post infection, IL-1β (B) and IL-6 (D) levels in the peritoneal lavage were measured by ELISA. Data are shown as mean ± SEM. H. Photographs (24 h) of Balb/c mice infected with 2.5 × 108 E. coli BL21 receiving PBS or rPPE18 (100 µg). I. E. coli BL21 bacteria were grown to log phase (A600 = 0.6) in the presence of 3 µg/ml of either BSA or rPPE18 or PBS as vehicle control. Prior to treatment, both BSA and rPPE18 were made endotoxin free by incubation with polymyxin B-agarose. The cultures were set up in 200 µl volume of LB (Luria Bertani) medium in 96 well plate and growth was monitored by reading absorbance at every 15 min for 6 h with a Spectramax M5 Series Multi-Mode Microplate Reader (Molecular Devices,

Sunnyvale, CA, USA). Data shown are mean ± SEM of four replicates. A p < 0.005 300

200

100 ALT U/L

0

E. coli

E. coli

PBS PBS rPPE18 B

C

Supplemental Figure 3. Pre-treatment with rPPE18 reduces organ damage in high dose infected septic mice. Balb/c mice were given either 100 µg of rPPE18 (n = 10) or an equivalent volume of PBS (n = 10) intraperitoneally prior to infection with 5.0 × 10 8 E. coli BL21 CFU. At 3 h post-infection ALT levels were measured in sera (A). The control group (n = 5) received PBS alone. Mice surviving after 20 h of infection were sacrificed and liver (B) and spleen (C) sections were prepared and stained with hematoxylin and eosin. Photographs of representative sections visualized at 100x magnification are shown. A B

15 20 p < 0.05 p < 0.05

15 10

10 IL-6 ng/ml IL-6

IL-12 p70 ng/ml p70 IL-12 5 5

0 0

E. coli E. coli

Supplemental Figure 4. Therapeutic administration of rPPE18 reduces IL-12 p70 and IL-6 levels in mice infected with E. coli

BL21. Balb/c mice were infected with 2.5 × 10 8 E. coli BL21 CFU by intraperitoneal injection and 1 h later 100 µg of rPPE18 or an

equivalent volume of PBS was injected intraperitoneally. Levels of IL-12 p70 (A) and IL-6 (B) in sera were measured by ELISA. Data

are shown as mean ± SEM.