**MICROBIAL MODULATION OF HOST APOPTOSIS AND PYROPTOSIS**

# **Topic Editors Yongqun He and Amal O. Amer**

CELLULAR AND INFECTION MICROBIOLOGY

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**ISSN** 1664-8714 **ISBN** 978-2-88919-280-9 **DOI** 10.3389/978-2-88919-280-9

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## **MICROBIAL MODULATION OF HOST APOPTOSIS AND PYROPTOSIS**

Topic Editors: **Yongqun He,** University of Michigan Medical School, USA **Amal O. Amer,** The Ohio State University, USA

Panels in the figure summarize major findings and reviews in the Issue describing "Microbial Modulation of Host Apoptosis and Pyroptosis":

Due to space limitation, this figure does not include all the details from the above listed papers, and it does not include many findings and reviews reported in other papers authored by Pei et al, Marriott, and Demehri et al.

This image was prepared by the editors Yongqun He and Amal O. Amer

Infectious disease is the result of an interactive relationship between a microbial pathogen and its host. In this interaction both the host and the pathogen attempt to manipulate each other using a complex network to maximize their respective survival probabilities. Programmed host cell death is a direct outcome of host-pathogen interaction and may benefit host or pathogen depending on microbial pathogenesis. Apoptosis and pyroptosis are two common programmed cell death types induced by various microbial infections. Apoptosis is non-inflammatory programmed cell death and can be triggered through intrinsic or extrinsic pathways and with or without the contribution of mitochondria. Pyroptosis is an inflammatory cell death and is typically triggered by caspase-1 after its activation by various inflammasomes. However, some non-canonical caspase-1-independent proinflammatory cell death phenomena have been reported.

Microbial pathogens are able to modulate host apoptosis and pyroptosis through different triggers and pathways. The promotion and inhibition of host apoptosis and pyroptosis vary and depend on the microbe types, virulence, and phenotypes. For example, virulent pathogens and attenuated vaccine strains may use different pathways to modulate host cell death. Specific microbial genes may be responsible for the modulation of host cell death. Different host cells, including macrophages, dendritic cells, and T cells, can undergo apoptosis and pyroptosis after microbial infections. The pathways of host apoptosis and pyroptosis induced by different microbes may also differ. Different methods can be used to study the interaction between microbes and host cell death system.

The articles included in this E-book report the cutting edge findings in the areas of microbial modulation of host apoptosis, pyroptosis and inflammasome.

# Table of Contents

## Microbial modulation of host apoptosis and pyroptosis

#### *Yongqun He1 \* and Amal O. Amer 2 \**

*1 Unit for Laboratory Animal Medicine, Department of Microbiology and Immunology, Center for Computational Medicine and Bioinformatics, Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA*

*2 Department of Microbial Infection and Immunity, Department of Internal Medicine, Center for Microbial Interface Biology, Ohio State University, Columbus, OH, USA*

*\*Correspondence<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### *Edited and reviewed by:*

*Yousef Abu Kwaik, University of Louisville School of Medicine, USA*

**Keywords: apoptosis, pyroptosis, microbial infection, caspase, inflammasome,** *Brucella***,** *Legionella pneumophila***,** *Mycobacterium tuberculosis*

Apoptosis and pyroptosis are two common programmed cell death types induced by various microbial infections. Apoptosis is non-inflammatory programmed cell death and can be triggered through intrinsic or extrinsic pathways and with or without the contribution of mitochondria. Pyroptosis is an inflammatory cell death and is typically triggered by caspase-1 after its activation by various inflammasomes. Non-canonical caspase-11-mediated pyroptosis has been identified. A NLRP3 (cryopyrin)-dependent but casepase-1-independent proinflammatory necrosis called pyronecrosis (Willingham et al., 2007), and a caspase-2 dependent but caspase-1-independent proinflammatory cell death (Chen et al., 2011) have also been reported. Microbial pathogens are able to modulate host apoptosis, pyroptosis, and inflammasomes through different triggers and pathways. The promotion and inhibition of host cell death vary and depend on the microbe types, virulence, and phenotypes.

In this Special Research Topics issue, recent advances in microbial modulation of host programmed cell death, with a special focus on apoptosis and pyroptosis, were captured in a total of 11 research and review articles. The special issue includes three Original Research Articles, five Review Articles, and three Mini Review Articles. Two articles were published for each of the three pathogens: *Brucella* spp. (Bronner et al., 2013; Pei et al., 2014), *Legionella pneumophila* (Abu Khweek et al., 2013; Casson and Shin, 2013), and *Mycobacterium tuberculosis* (Aguilo et al., 2013; Parandhaman and Narayanan, 2014). Modulation of host immune defenses by *Aeromonas* and *Yersinia* species is introduced in Rosenzweig and Chopra (2013). While (Cunha and Zamboni, 2013) summarizes the subversion of inflammasome activation and pyroptosis by eight pathogenic bacteria, (Malireddi and Kanneganti, 2013) introduces the role of type I interferons in inflammasome activation and cell death induced by microbial infections. The apoptosis-associated uncoupling of bone formation and resorption in osteomyelitis is reviewed in Marriott (2013). The intestinal epithelial cell apoptosis in the setting of altered microbiota with enteral nutrient deprivation is reviewed in Demehri et al. (2013).

*Brucella* causes brucellosis, one of the most common zoonotic diseases in the world in humans and a variety of animal species. The *Brucella*-macrophage interaction is critical to *Brucella* virulence. Virulent smooth *Brucella* strains inhibit macrophage cell death. This is an important strategy employed by several intracellular pathogens to maintain the survival of the eukaryotic cell that represents its niche. Many attenuated rough *Brucella* strains induce macrophage cell death. The Original Research Article (Pei et al., 2014) demonstrates that after smooth *Brucella* invade and replicate inside host macrophages, some smooth bacteria can automatically dissociate into rough mutants that can then cause the macrophage cytotoxicity. The cytotoxicity of infected macrophages is critical for *Brucella* egress and dissemination. The macrophage necrotic cell death also induces inflammatory responses and recruits more macrophages to the infection site.

The rough attenuated *B. abortus* vaccine strain RB51 was found to induce caspase-2-mediated but caspase-1-independent apoptotic and necrotic cell death (Chen and He, 2009). Original Research Article (Bronner et al., 2013) from this special issue further illustrates this mechanism. In RB51-infected macrophages, caspase-2 regulates many genes and several cell death pathways: (i) proapoptotic caspases-3 and -8 activation; (ii) mitochondrial cytochrome *c* release and TNFα production; (iii) caspase-1 and IL-1β production driven by caspase-2-mediated mitochondrial dysfunction. Unlike *S. typhimurium*-induced caspase-1-mediated pyroptosis, RB51-induced pore formation does not contribute to RB51-induced proinflammatory cell death. Therefore, caspase-2 appears to act as a "master regulator" that regulates various genes and pathways and induces a hybrid cell death with features of both apoptosis and pyroptosis. The caspase-2-mediated cell death was also conserved in macrophages treated with cellular stress inducers including etoposide, naphthalene, or anti-Fas (Bronner et al., 2013).

Interesting study by Abu Khweek compared the innate immune response of planktonic and biofilm-derived *L. pneumophila*. *L. pneumophila*, the causative agent of Legionnaire's disease, replicates inside macrophages to establish infection. In the Original Research Article (Abu Khweek et al., 2013), the authors demonstrated that compared to planktonic *L. pneumophila*, biofilm-derived *L. pneumophila* (i) replicate more in murine macrophages, (ii) lacks flagellin expression, (iii) do not activate caspase-1 or -7, (iv) trigger less cell death, and (v) are mostly enclosed in vacuoles that do not fuse with lysosomes. Therefore, biofilm-derived *L. pneumophila* which closely reproduces the natural mode of the bacterial infection in human is able to evade the innate immune response in murine macrophages.

The canonical pyroptosis is triggered by the inflammasome, a multi-protein complex assembled in the cytosol to activate caspase-1. A non-canonical inflammasome activates caspase-11 and also leads to pro-inflammatory cell death (Kayagaki et al., 2011). Independently of the inflammasome, caspase-11 promotes the fusion of the *L. pneumophila*-containing vacuole with the lysosome (Akhter et al., 2012). The diverse roles of caspase-11 and routes of activation are described in the mini-review (Casson and Shin, 2013) *L. pneumophila* triggers canonical caspase-1 dependent inflammasome activation through one of two pathways: (i) Type 4 secretion system (T4SS)-regulated flagellin, NAIP5, and NLRC4; (ii) host ASC and NLRP3, and a *L. pneumophila*-derived unknown signal. Molecular details on caspase-11 activation in *L. pneumophila*-infected macrophages remain unclear. Interestingly, the inflammasome pathway appears to cross talk with and autophagy, another immune response (Casson and Shin, 2013).

*Mycobacterium tuberculosis,* another professional intracellular pathogen in this issue, also manipulates cell death. Conflicting results have been reported to support inhibition or induction of apoptosis as a virulence mechanism employed by mycobacteria. This elegant review article (Aguilo et al., 2013), summarizes the evidences showing that ESX-1-induced apoptosis during mycobacterial infection contributes to bacterial virulence. The ESX-1 secretion system regulates the exportation of ESAT-6, a major virulence factor whose secretion is essential for *M. tuberculosis*-induced apoptosis. ESAT-6 appears to trigger the mitochondrial apoptotic pathway through ER-stress activation. ESX-1 dependent apoptosis supports cell-to-cell colonization and bacterial spread. Highly apoptogenic *M. tuberculosis nuoG* mutant showed higher cell-to-cell spread and increased antigen crosspresentation favoring the host. It is evident that apoptosis may benefit the host or mycobacterial pathogen according to different experimental conditions (Aguilo et al., 2013).

The well rounded report (Parandhaman and Narayanan, 2014) summarizes more than 10 different cell death modalities involving *M. tuberculosis*. The paper also reviews how PknE, one of 11 mycobacterial serine/threonine protein kinases, inhibits apoptosis and benefits the bacterial survival.

This issue also comprises a paper (Rosenzweig and Chopra, 2013) that describes toxins secreted by pathogenic *Yersiniae* and most *Aeromonas* species that modulate infected host cell death. The T3SS effector *Yersinia* outer membrane protein J (YopJ) is an acetyltransferase that disrupts MAPK and NF-κB signaling pathways to favor apoptosis and pyroptosis induction. Similarly, *Aeromonas* hydrophila AexU protein induces apoptosis by targeting NF-κB signaling. Additionally *Aeromonas* includes T2- and T6SS effectors that further modulate host immune responses to promote bacterial virulence (Rosenzweig and Chopra, 2013).

The nice paper by Zamboni's group (Cunha and Zamboni, 2013) first reviews different types of inflammasomes that activate caspase-1 (via NLRC4, AIM2, or NLRP3) or caspase-11 and then lead to pyroptosis. These host inflammasomes and pyroptosis pathways can be targeted by microbial factors released via T3SS/T4SS or other mechanisms in different pathogens. This paper reviews the mechanisms employed by eight bacterial species to evade inflammasome activation and pyroptosis induction. These bacteria include *Chlamydia trachomatis*, *Coxiella burnetii*, *Francisella tularensis*, *Legionella pneumophila, Pseudomonas aeruginosa*, *Shigella flexneri*, *Vibrio parahaemolyticus*, and *Yersinia* spp. (Cunha and Zamboni, 2013).

On the host side, the review by Kanneganti's group (Malireddi and Kanneganti, 2013) in this issue introduces the role of type I interferons in inflammasome activation and cell death during infections of five intracellular, four extracellular bacteria, viruses, and fungi.

How cell death can lead to human disease conditions is well described by the review paper (Marriott, 2013) that focuses on osteomyelitis, a severe infection of bone caused by *S. aureus* and *Salmonella* spp. Osteomyelitis is often associated with bone resorption and progressive inflammatory destruction. In the paper Marriott describes the mechanisms underlying the destruction of bone tissue, with a focus on the apoptosis-associated uncoupling of bone formation and resorption in osteomyelitis. Different microbial virulence factors, host response genes and pathways, and their interactions during the formation of osteomyelitis are introduced.

It has been well established that pathogens modulate apoptosis and pyroptosis, but what about microbiota? The paper (Demehri et al., 2013) in this issue introduces a shift in our understanding of intestinal microbiota such as Gram-negative Proteobacteria after enteral nutrient deprivation. The altered microbiota setting leads to increased intestinal proinflammatory cytokines, decreased epithelial cell proliferation, and increased epithelial cell apoptosis. These eventually cause the loss of epithelial barrier function.

The cover image of this E-book summarizes the key findings reported in the original research, review, or mini-review articles included in this e-book.

As briefly introduced above, this special Research Topic issue covers a broad range of cases and reviews demonstrating the modulation of host cell death pathways by different bacterial pathogens and resident microbiota. While huge progress has been made in the past decades, many challenging questions still remain.

## **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 June 2014; accepted: 03 June 2014; published online: 19 June 2014. Citation: He Y and Amer AO (2014) Microbial modulation of host apoptosis and pyroptosis. Front. Cell. Infect. Microbiol. 4:83. doi: 10.3389/fcimb.2014.00083 This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2014 He and Amer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Biofilm-derived *Legionella pneumophila* evades the innate immune response in macrophages

*Arwa Abu Khweek1, Natalia S. Fernández Dávila1, Kyle Caution1, Anwari Akhter 1, Basant A. Abdulrahman1,2, Mia Tazi 1, Hoda Hassan1, Laura A. Novotny3, Lauren O. Bakaletz 1,3 and Amal O. Amer 1 \**

*1 Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, College of Medicine, The Ohio State University, Columbus, OH, USA*

*2 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University, Cairo, Egypt*

*3 Center for Microbial Pathogenesis, Nationwide Children's Hospital, Columbus, OH, USA*

#### *Edited by:*

*Yongqun Oliver "He", University of Michigan School of Medicine, USA*

#### *Reviewed by:*

*Sunny Shin, University of Pennsylvania, USA Cyril Guyard, Public Health Ontario, Canada*

#### *\*Correspondence:*

*Amal O. Amer, Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, Dorothy Heart and Lung Research Institute, Ohio State University, Biological Research Tower, 460 W 12th Ave., Room 706, Columbus, OH 43210, USA. e-mail<EMAIL_ADDRESS>
*Legionella pneumophila*, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is *L. pneumophila* biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. *L. pneumophila* infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic *L. pneumophila* elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic *L. pneumophila* and murine or human macrophages have been studied for years, yet the interface between biofilm-derived *L. pneumophila* and macrophages has not been explored. Here, we demonstrate that biofilm-derived *L. pneumophila* replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic *L. pneumophila*, biofilm-derived *L. pneumophila* lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic *L. pneumophila* is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived *L. pneumophila* and closely reproduces the natural mode of infection in human.

#### **Keywords: biofilm, inflammasome, flagellin, caspase-1,** *Legionella pneumophila***, innate immunity**

### **INTRODUCTION**

*Legionella pneumophila* (*L. pneumophila*) is a Gram negative facultative bacterium with fastidious growth requirements. Although *Legionella* exists as free-living planktonic forms in the environment, they are more commonly found as intracellular parasites of protozoans such as *Acanthamoeba* spp., *Hartmannella* spp., and *Tetrahymena* spp. (Atlas, 1999; Brown and Barker, 1999) and as inhabitants of mixed-community biofilms (Rogers et al., 1994; Lau and Ashbolt, 2009). Replication of *L. pneumophila* within amoeba is utilized as a survival strategy to overcome the low-nutrient environment and increases the resistance to disinfectant (Lau and Ashbolt, 2009). This opportunistic pathogen most often thrives in bacterial communities encased in extracellular polymeric matrix known as biofilm (Costerton et al., 1978; Donlan et al., 2005). Biofilms have been recognized as one of the most important factors of survival and proliferation of *L. pneumophila* in warm, humid environments like showers, air conditioners, and spa baths (Fraser et al., 1979; Fliermans et al., 1981; Sethi and Brandis, 1983; Spitalny et al., 1984; Abu Kwaik et al., 1993; Lettinga et al., 2002). These communities have been identified as a causative source of infection in susceptible hosts

who inhale aerosols of contaminated water containing *L. pneumophila*. In the human lung environment, *L. pneumophila* replicates exponentially within alveolar macrophages prior to lysing the host cell and invading other macrophages causing a type of walking pneumonia called Legionnaire's disease or Legionellosis (Horwitz and Silverstein, 1980; Harb and Abu Kwaik, 2000). Legionellosis has two clinically distinct forms: Legionnaires' disease, a severe type of infection, which includes pneumonia and Pontiac fever, a milder self-limiting illness (Lau and Ashbolt, 2009). Approximately 20,000 cases of Legionnaire's disease are reported yearly in the US with no person-to-person transmission (Marston et al., 1997). Thus, using biofilm-derived *L. pneumophila* to study the innate immune response to infection recapitulates natural mode of infection in human.

The murine innate immune response to planktonic *L. pneumophila* has been studied extensively. Nlrc4 and Naip5 detect flagellin monomers in the host cytosol in a process that is dependent upon a functional bacterial type IV secretion system. The sensing of contaminating molecules of flagellin promotes the formation of a multi-protein complex called the inflammasome. Within the inflammasome, caspase-7 is activated downstream of caspase-1 which results in bacterial restriction via fusion of *L. pneumophila*-containing vacuoles with lysosomes (Coers et al., 2000; Akhter et al., 2009; Amer, 2010). Conversely, human monocytes do not activate this response upon *L. pneumophila* infection and phagosomes containing *L. pneumophila* evade fusion with the lysosome allowing bacterial replication (Roy, 2002; Isberg et al., 2009).

Here we demonstrate that biofilm-derived *L. pneumophila* replicates significantly more than planktonic *L. pneumophila* in murine macrophages due to diminished flagellin expression. Biofilm-derived *L. pneumophila* does not activate caspase-1 or caspase-7, evades fusion with lysosomes, and promotes less cell death. Taken together, our study characterizes the innate immune response to biofilm-derived *L. pneumophila* in murine and human macrophages.

### **METHODS**

#### **BACTERIAL STRAINS**

*L. pneumophila* strain JR32 a wild-type (WT) strain and *flaA* mutant is deficient in flagellin, were kindly provided by Dr. Howard Shuman, University of Chicago. The *dotA* mutant, a JR32-derived strain defective in the Dot/Icm Type IV secretion system was kindly provided by Dr. Craig Roy, Yale School of Medicine. *L. pneumophila* expressing green fluorescent protein (GFP) was used for microscopy.

#### *L. pneumophila* **GROWTH**

*L. pneumophila* strains were grown on buffered charcoal yeast extract (BCYE) plates at 37◦C. Three days later, the bacteria were resuspended in 5 ml of *L. pneumophila* medium (BYE) with additives (ferric nitrate, L-cysteine, thymidine) and vortexed 100× at high speed. For biofilm formation, a bacterial suspension with an optical density (OD) at 600 nm of 3.5 was diluted to 1:2500 in supplemented broth and 200 μl of this suspension was inoculated into each well of an 8-well chamber slide (Thermo Scientific Lab Tek chambered coverglass with cover #155411 and/or #177402). Slides were incubated at 37◦C, 5% CO2 incubator with humidified atmosphere without shaking. Biofilms were fed by delivery of fresh medium to one side of the chamber slide well every 24 h for 6 days using 100μl of *L. pneumophila* medium. The OD values were 3.4–3.6 and 3.8–4 for the JR32 and *dotA* mutant, respectively. *L. pneumophila* was grown for planktonic culture as previously described (Amer et al., 2006; Akhter et al., 2009, 2012).

#### **MACROPHAGE INFECTION**

C57BL/6 mice were purchased from Jackson laboratory. Bone marrow derived macrophages (BMDMs) were prepared from the femurs of 6 to 8-week-old mice as previously described (Akhter et al., 2009, 2012). Isolation and preparation of the human monocyte-derived macrophages (hMDMs) from peripheral blood was carried out as previously described (Santic et al., 2005; Al-Khodor et al., 2008). Planktonic infections were used from post-exponential cultures as previously described (Amer et al., 2006; Akhter et al., 2009). Infection from biofilm derived *L. pneumophila* was carried as follows. Briefly, on day seven, the media was aspirated and transferred to a 50 mL tube. Biofilms were scraped from the chamber slide wells. Chambers were washed 2× with 200μl of fresh LP medium with the previously mentioned additives and drained into the 50 ml tube. The collected biofilms were vortexed 100× and the OD at 600 nm of the collected suspension was used to calculate the desired multiplicity of infection. Equivalent inocula of planktonic bacteria were used for infection (MOIs).

*L. pneumophila* is an intracellular pathogen that replicates only within eukaryotic cells since the culture media do not contain required nutrients such as iron and cysteine.

#### **CONFOCAL LASER SCANNING MICROSCOPE VISUALIZATION**

On day seven, biofilms were washed gently with 200μl of sterile saline (0.9% sodium chloride) (Hospira 0409-4888-10), and stained using the Live/Dead BacLight Bacterial Viability Kit (Invitrogen #7007) for 15 min at room temperature protected from the light. Wells were washed 2× with sterile saline and 200μl of 10% formalin was added for 24 h to fix the biofilm and stored at room temperature protected from the light with before it was visualized using inverted confocal Zeiss LSM 510 META microscope with a 63× water objective. Z-stacks were captured every 1μm.

#### **ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)**

*L. pneumophila* JR32 and *dotA* strains from post-exponential planktonic and biofilm cultures were used to infect murine macrophages for 24 h. Supernatants were collected and centrifuged at 1200 rpm for 10 min and stored at −80◦C as previously described (Abdulrahman et al., 2011). The plates were coated with primary antibody for IL-1β and ELISA were performed according to the manufacture specifications (R&D) (Abdulrahman et al., 2011).

#### **WESTERN BLOT**

Macrophage lysates were prepared following infection with either planktonic or biofilm JR32 or *dotA* mutant and immunoblotted with caspase-1, caspase-7, or β-actin antibodies (caspase-1, 1:3000; caspase-7, 1:300). Blots were washed and the corresponding secondary antibody was added. For flagellin detection by western blot, one OD of bacterial culture was pelleted and resuspended in SDS-containing sample buffer from planktonic or biofilm grown bacteria. Eighteen μl were loaded on 12% SDS-PAGE gel. The blot was probed with flagellin antibody (1:100) kindly provided by Dr. Howard Shuman, University of Chicago, followed by the secondary antibody, donkey anti-rabbit (1:5000). Blots were developed after adding ECL Western Blotting Detection Reagent (GE Healthcare Amersham).

#### **MACROPHAGE CYTOTOXICITY ASSAY**

Percentage of macrophage necrosis was determined by measuring the release of host cell cytoplasmic lactate dehydrogenase (LDH) using the cytotoxicity detection kit (Roche Applied Science) to the specification of the manufacturer. BMDMs were infected with JR32 or the *dotA* mutant from either planktonic or biofilm culture for 4 or 24 h at an MOI of 0.5. Supernatants were collected and LDH release was calculated as previously described (Abdulrahman et al., 2011).

#### *L. pneumophila* **COLOCALIZATION WITH LYSOTRACKER**

*L. pneumophila* JR32 from post-exponential planktonic or biofilm cultures were used to infect macrophages plated in 24-well plates containing sterilized coverslips. Lysotracker red (1:500) was added before fixation and 4 , 6-diamidino-2-phenylindole (DAPI) was added after fixation. Coverslips were mounted on slides and viewed using the Olympus Flow View FV10i CLSM. Three hundred bacteria were counted from 2 coverslips for each condition.

#### **CONTACT-DEPENDENT HEMOLYSIS**

Sheep RBCs (sRBCs) were diluted in RPMI, and washed 3× by centrifugation for 10 min at 2000× g until the supernatant did not show any signs of hemolysis; the cells were counted using a hemo-cytometer chamber. Reactions were set up in a final volume of 1 ml with a final concentration of 1 × 107 sRBCs/ml. The sRBCs were incubated with the planktonic or biofilm bacteria at an MOI of 20 and RBC lysis was determined as previously described (Kirby et al., 1998; Alli et al., 2000).

#### **SCANNING ELECTRON MICROSCOPY (SEM)**

*L. pneumophila* strains were grown on 12-well plate coverslips for seven days. On the seventh day, the medium was aspirated and the coverslips were washed with 1× DPBS. Coverslips were fixed with 2.5% gluteraldehyde in 0.1 M phosphate buffer pH 7.4, processed and viewed by SEM.

### **STATISTICAL ANALYSIS**

Experiments were performed 2–3 independent times each in triplicate or quadruplicate and yielded similar results. Comparisons of groups for statistical significance were performed using Student's tow tailed *t*-test. *P*-values ≤ was considered significant.

## **RESULTS**

#### **THE Dot/Icm TYPE IV SECRETION SYSTEM PROMOTES ROBUST** *L. pneumophila* **BIOFILM FORMATION**

To reproduce biofilm formation *in vitro*, WT *L. pneumophila* (JR32) and *dotA* mutant were grown for 7 days at 37◦C on 5% CO2 in 8-well chambered coverslips and fed with *L. pneumophila* BYE media every 24 h. On the seventh day, biofilms were stained with Live/Dead stain then observed by confocal microscopy. *L. pneumophila* JR32 produced a thick biofilm with a maximum height of ∼120μm and exhibited filamentous structures.

The *dotA* mutant, which lacks a functional type IV secretion system, produced a thinner and less filamentous biofilm (**Figure 1A**) that was 60μm high (**Figure 1B**). The homogenous green color, with lack of red coloring indicates that the constituent bacteria are viable. This result suggests that a functional Dot/Icm type IV secretion system promotes biofilm formation.

To confirm that *L. pneumophila* is able to form biofilm *in vitro* and this process is dependent upon type IV secretion system, we

examined biofilm formation using scanning electron microscopy (SEM). Our data showed that JR32 strain formed a robust biofilm with characteristic towers whereas the *dotA* mutant failed to do so (**Figure 1C**). These data confirm that robust biofilm formation requires type IV secretion system.

The pore forming activity of *L. pneumophila* has been shown to contribute to macrophage cytotoxicity and requires a functional type IV secretion system (Kirby et al., 1998; Alli et al., 2000). To examine whether biofilm-derived *L. pneumophila* exhibit pore-forming activity, contact-dependent hemolysis of sheep red blood cells (RBCs) was performed (Kirby et al., 1998). Triton-X100 and heat-killed bacteria were used as positive and negative controls, respectively. The *flaA* mutant lacking flagellin and expresses a functional type IV secretion system was also examined (**Figure 1D**). Biofilm-derived and planktonic *L. pneumophila* and *flaA* mutant were capable of lysing the RBCs, suggesting that biofilm-derived *L. pneumophila* exhibit a functional type IV secretion system.

#### **BIOFILM-DERIVED** *L. pneumophila* **REPLICATES SIGNIFICANTLY MORE INTRACELLULARLY AND INDUCES LESS MURINE MACROPHAGE DEATH THAN THE PLANKTONIC BACTERIA**

WT murine macrophages are restrictive to planktonic *L. pneumophila* replication. However, the murine macrophages response to biofilm-derived *L. pneumophila* is not known. Therefore, we examined the intracellular replication of biofilm-derived *L. pneumophila*. In contrast to planktonic bacteria, the biofilmderived bacteria replicated significantly as indicated by the colony forming units (CFUs) over time (48–96 h) (**Figure 2A**). Macrophages phagocytozed similar numbers of biofilm-derived and planktonic *L. pneumophila* as demonstrated by the 1 h CFU counts.

Premature cell death restricts *L. pneumophila* replication within murine macrophages (Akhter et al., 2009, 2012). Macrophage death can be detected by measuring the enzymatic activity of lactate dehydrogensae (LDH) released from dead cells using a LDH cytotoxicity assay. Our data demonstrated

**FIGURE 2 | Biofilm-derived** *L. pneumophila* **replicates significantly more and induces less murine macrophages death than the planktonic bacteria. (A)** BMDMs were infected with planktonic or biofilm-derived *L. pneumophila* at an MOI of 0.5. CFUs were scored at 1, 24, 48, 72, and 96 h. Data are presented as mean ± SD of two independent experiments each performed in duplicates. Asterisks indicate significant differences (∗∗∗*P <* 0*.*001). BMDMs were not infected (NT) or infected with *L. pneumophila* JR32 (planktonic or biofilm) or the *dotA* mutant at an MOI of 0.5 for **(B)** 4 or **(C)** 24 h. The fold change in LDH release was measured from the overall population of macrophages. Data are

presented as means ± SD of two independent experiments each performed in quadruplicates. Asterisks indicate significant differences (∗∗*P <* 0*.*01). **(D)** The hMDMs were infected with *L. pneumophila* strain JR32 (planktonic or biofilm). CFUs were quantified at 1, 24, and 48 h post-infection. Data are representative as means ± SD of quintuplicate samples. **(E)** The hMDMs were not infected (NT) or infected with *L. pneumophila* JR32 (planktonic or biofilm), *dotA* or the *flaA* mutant at an MOI of 0.5 for 4 or 24 h. The fold change in LDH release was measured from the overall population of macrophages. Data are representative of mean ± SD of quadruplicate samples.

that macrophages infected with biofilm-derived *L. pneumophila* produced less LDH at 4 and 24 h post-infection when compared to those infected with planktonic bacteria (**Figures 2B**,**C**). Biofilm-derived and planktonic *dotA* mutants led to the same extent of macrophage death. These data suggest that biofilmderived *L. pneumophila* induced less cell death in murine macrophages than did planktonic-derived *L. pneumophila*.

#### **HUMAN MACROPHAGES ARE PERMISSIVE TO BIOFILM-DERIVED** *L. pneumophila* **AS THEY ARE TO PLANKTONIC CULTURES**

In contrast to murine macrophages, human monocytesderived macrophages (hMDMs) are permissive to planktonic *L. pneumophila* at least in part due to diminished caspase-1 and -7 activation (Horwitz, 1983; Abdelaziz et al., 2011a,b). Replication within macrophages is essential for establishing Legionnaire's pneumonia. Thus, we evaluated the intracellular growth of biofilm-derived *L. pneumophila* in hMDMs. The biofilm-derived *L. pneumophila* replicated similar to planktonic *L. pneumophila* in hMDMs (**Figure 2D**). As expected the *dotA* mutant did not replicate whereas *L. pneumophila* mutant lacking flagellin replicated the most (**Figure 2D**). This difference was not due to differential uptake since phagocytosis of all tested strains was similar as shown by the 1 h CFUs (**Figure 2D**).

Furthermore, we tested macrophage death by measuring percentage of LDH released after 24 h of infection. Planktonic and biofilm-derived *L. pneumophila* caused similar amount of LDH release from hMDMs while *dotA* mutant caused less cell death (**Figure 2E**). Human macrophages infected with the *flaA* mutant also released comparable amounts of LDH (**Figure 2E**). These data suggest that biofilm-derived *L. pneumophila* behave similarly to planktonic *L. pneumophila* in hMDMs.

#### **BIOFILM-DERIVED** *L. pneumophila* **AVOIDS CASPASE-1 AND -7 ACTIVATION IN MURINE MACROPHAGES DUE TO LACK OF FLAGELLIN EXPRESSION**

Upon detection of bacterial flagellin by Nlrc4 and Naip5, WT murine macrophages restrict planktonic *L. pneumophila* replication via caspase-1 and -7 activation. Caspase-7 promotes the fusion of the *L. pneumophila*-containing vacuole with the lysosome and bacterial degradation whereas caspase-1 contributes to pyroptosis and IL-1β release (Akhter et al., 2009, 2012). Because biofilm-derived *L. pneumophila* replicate more efficiently in murine macrophages and exhibited less cell death when compared to planktonic, we tested whether mouse macrophages activated caspase-1 in response to biofilm-derived bacteria. The *dotA* and *flaA* mutants were used as negative controls since they both avoid caspase-1 activation. In contrast to planktonic bacteria, biofilm-derived *L. pneumophila* did not promote caspase-1 activation as denoted by the detection of the cleaved active band by western blot (**Figure 3A**). These data indicate that murine macrophages respond to biofilm-derived *L. pneumophila* differentially than to planktonic *L. pneumophila*.

IL-1β maturation is promoted by active caspase-1 in WT macrophages infected with planktonic *L. pneumophila*. Therefore, we tested IL-1β release in culture supernatants from murine macrophages infected with planktonic, biofilm-derived *L. pneumophila* and the *dotA* mutant. Our data demonstrate that murine macrophages infected with biofilm-derived bacteria released

30% less IL-1β compared to that released after planktonic *L. pneumophila* infection (**Figure 3B**). This result indicates that the inflammatory response to biofilm-derived *L. pneumophila* in murine macrophages is less than that elicited in response to planktonic bacteria.

During infection of planktonic *L. pneumophila*, murine macrophages activate caspase-7 via the inflammasome complex contributing to bacterial restriction (Akhter et al., 2009, 2012), but this response has not been characterized in biofilmderived *L. pneumophila*. Therefore, we tested caspase-7 activation of murine macrophages in response to biofilm-derived and planktonic *L. pneumophila*. In contrast to planktonic bacteria, biofilm-derived *L. pneumophila* did not promote caspase-7 cleavage (**Figure 3C**), indicating that biofilm-derived bacteria do not elicit caspase-7 activation, thereby allowing them to evade a restrictive mechanism employed by murine macrophages.

Flagellin mediates restriction of *L. pneumophila* in murine macrophages and the *flaA* mutant has been shown to replicate significantly more than the parent strain (Amer et al., 2006). Since biofilm-derived bacteria replicated significantly in murine macrophages and failed to activate caspase-1 or 7, we hypothesized that biofilm-derived bacteria down regulates flagellin expression. Western blot analysis of bacterial lysates using specific flagellin antibodies demonstrated that biofilm-derived bacteria diminished flagellin expression compared to planktonic bacteria (**Figure 3D**). Collectively, these results indicate that biofilm *L. pneumophila* do not activate the inflammasome because of lack of flagellin expression compared to planktonic *L. pneumophila*.

#### **PHAGOSOMES CONTAINING BIOFILM-DERIVED** *L. pneumophila* **EVADE FUSION WITH THE LYSOSOMES**

In murine macrophages, *L. pneumophila* replication is restricted by caspase-1 and -7 activation that result in phagosomelysosome fusion, promoting bacterial degradation (Akhter et al., 2009, 2012). We examined the colocalization of planktonic and biofilm-derived bacteria with lysosomes at 1 h post-infection. Approximately 55% of biofilm-derived *L. pneumophila* resided in lysosomes (**Figures 4A**,**B**). Yet, 72% of planktonic bacteria resided in lysosomes. These results suggests that significantly more biofilm-derived *L. pneumophila* evade lysosomal degradation in macrophages allowing the bacteria to survive within the host and replicate as indicated by increased CFUs in (**Figure 4A**).

#### **DISCUSSION**

Legionnaire's disease is a severe pneumonia that infects the elderly and the immune compromised. There is no human to human transmission whereas infection occurs by inhalation of contaminated droplets from biofilms lining air conditioners and fresh water fountains. A biofilm is a highly-organized, multicellular community affixed to an inert or biological surface and is the preferred lifestyle of most bacteria. Bacterial

**significantly evade fusion with lysosomes. (A)** Representative images of WT BMDMs infected for 1h with JR32 planktonic or biofilm. Nuclei are stained blue with DAPI and *L. pneumophila* stained green with *L. pneumophila*-specific antibody. Lyso-tracker red was used to stain acidified lysosomes. White arrows colocalization of *L. pneumophila* with lysotracker. Images were captured with the 60× objective and magnified 3×, scale bar = 10μm. Data are presented as means ± SD of three independent experiments each performed in triplicates. Asterisks indicate significant difference (∗∗*P <* 0*.*01).

populations within a biofilm, as opposed to their planktonic counterparts, are highly resistant to eradication (Flemming and Wingender, 2010). While some pathogens form biofilms within infected organs to resist phagocytosis and immune responses, others such as *L. pneumophila* form biofilms in nature and in medical and dental devices to serve as sources of outbreaks of Legionnaire's disease. In this case, the biofilm setting serves as niche where the bacterium is recalcitrant to most antibacterial agents (Rogers et al., 1994; Brown and Barker, 1999; Lau and Ashbolt, 2009).

In this study we reproduced *L. pneumophila* biofilm formation *in vitro*. Since infection occurs through the inhalation of *L. pneumophila*-containing droplets, we also simulated the dispersion of biofilm by vortex. Because once inhaled, the organism adapts an intracellular niche within macrophages, we examined the behavior of biofilm-derived *L. pneumophila* within murine and human macrophages.

We showed that biofilm-derived bacteria are more filamentous than planktonic bacteria (**Figure 1A**). This characteristic may be due to adaptation of the bacteria to a harsh environment providing an energy saving benefit for survival. Alternatively, the filamentous multinucleated characteristic may facilitate rapid division into planktonic form when encountering a parasitic host (Taylor et al., 2009). Notably, macrophages derived from mice and human donors phagocytozed equivalent numbers of biofilm-derived and planktonic organisms as demonstrated at 1 h post infection. Therefore, the filamentous phenotype of *L. pneumophila* within the biofilm did not impact their phagocytosis.

We demonstrated that robust biofilm formation by *L. pneumophila* is promoted by the DotA type IV secretion system. This finding corroborates several studies demonstrating the contribution of secreted bacterial molecules necessary for biofilm formation (Taylor et al., 2009). Secreted bacterial factors are utilized for quorum-sensing, biofilm building, and twitching motility during biofilm formation (Taylor et al., 2009). Therefore, identification and characterization of bacterial factors that are required for *L. pneumophila* biofilm formation can be targeted to prevent or eradicate biofilm formation and transmission of Legionnaires' disease in medical and industrial settings. Furthermore, *L. pneumophila* embedded within multispecies biofilms may respond to signal molecules produced by other bacteria promoting us to rethink current *L. pneumophila* research paradigms (Taylor et al., 2009).

Although the innate immune response of macrophages to planktonic *L. pneumophila* is under investigation by many scientists, the macrophage response to biofilm-derived *L. pneumophila* is yet to be elucidated. In this study, we found that biofilmderived *L. pneumophila* replicate significantly more in murine macrophages than do planktonic bacteria. This result suggests that studies employing planktonic *L. pneumophila* in murine macrophages may not recapitulate the biofilm-derived mode of infection in humans. Yet, hMDM responded similarly to biofilmderived and planktonic *L. pneumophila* establishing more validation for human based studies whether using planktonic or biofilm-derived *L. pneumophila*.

For an unidentified reason, planktonic flagellated *L. pneumophila* escapes detection by human NAIP and NLRC4 contributing to the permissiveness of human macrophages to the pathogen (Abdelaziz et al., 2011a,b; Ge et al., 2012). Studies by the Shao group indicated that human NAIP does not appear to respond to *L. pneumophila* flagellin (Ge et al., 2012). We also showed that human NLRC4 does not respond to flagellated *L. pneumophila* (Abdelaziz et al., 2011a,b). Yet, human NLRC4 activates caspase-1 in response to flagellated *Salmonella* (Abdelaziz et al., 2011b). Here we found that biofilm-derived *L. pneumophila* that seems to diminish flagellin expression replicates to the same extent as planktonic cultures in human macrophages. Since *L. pneumophila* flagellin detection is nonetheless avoided in human cells, it is plausible to expect that the *flaA* mutant would replicate to the same extent, however, this was not the case. The planktonic *flaA* mutant replicated more that the parent strain whether planktonic or biofilm-derived. This result suggests that biofilm-derived *L. pneumophila* may still express scarce amounts of flagellin contributing to its modest restriction via a caspase-1 independent mechanism. This remains to be elucidated by further studies.

Unlike planktonic *L. pneumophila*, biofilm-derived *L. pneumophila* do not activate caspase-1 and -7 in murine macrophages. The activation of caspase-1 and -7 promote the fusion of vacuoles containing *L. pneumophila* with lysosomes and bacterial degradation (Amer et al., 2006; Akhter et al., 2009; Franchi et al., 2009). Thus, our results suggest that biofilm derived *L. pneumophila* evades phagosome-lysosome fusion by avoiding caspase-1 and -7 activation. Macrophages infected with biofilm-derived bacteria exhibited significantly less IL-1β release and macrophage death compared to those infected with planktonic bacteria. IL-1β is a pro-inflammatory cytokine activated by caspase-1. Thus, by reducing IL-1β release, biofilm-derived *L. pneumophila* avoids major inflammatory responses and the recruitment of inflammatory cells to the infected lungs.

A better understanding of the innate immune response to biofilm-derived *L. pneumophila* will pave the way for the development of novel diagnosis and treatment strategies.

#### **AUTHOR CONTRIBUTIONS**

Arwa Abu Khweek designed and performed the experiments, analyzed the results, and wrote the manuscript. Natalia S. Fernández Dávila, Kyle Caution, Anwari Akhter, Basant A. Abdulrahman, Mia Tazi, Hoda Hassan, Laura A. Novotny, Lauren O. Bakaletz contributed to the performance of the experiments and editing of the manuscript. Amal O. Amer helped in the design of the experiments, interpretation of the results, and editing of the manuscript.

#### **ACKNOWLEDGMENTS**

Studies in Dr. Amal O. Amer's laboratory are supported by grant R01HL094586 and Public Health Preparedness for Infectious Diseases. Mia Tazi is supported by Bridge Funding provided by The College of Medicine and the Graduate School of The Ohio State University to support the training program for Microbial Interface Biology.

## **REFERENCES**

Legionella effector functioning in membrane trafficking. *Proc. Natl. Acad. Sci. U.S.A.* 109, 6193–6198.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 January 2013; accepted: 27 April 2013; published online: 27 May 2013.*

*Citation: Abu Khweek A, Fernández Dávila NS, Caution K, Akhter A, Abdulrahman BA, Tazi M, Hassan H, Novotny LA, Bakaletz LO and Amer AO (2013) Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages. Front. Cell. Infect. Microbiol. 3:18. doi: 10.3389/ fcimb.2013.00018*

*Copyright © 2013 Abu Khweek, Fernández Dávila, Caution, Akhter, Abdulrahman, Tazi, Hassan, Novotny, Bakaletz and Amer. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Modulation of host immune defenses by *Aeromonas* and *Yersinia* species: convergence on toxins secreted by various secretion systems

## *Jason A. Rosenzweig1,2\* and Ashok K. Chopra3,4,5,6\**

*1 Department of Biology, Center for Bionanotechnology and Environmental Research, Texas Southern University, Houston, TX, USA*

#### *Edited by:*

*Yongqun He, University of Michigan School of Medicine, USA*

#### *Reviewed by:*

*Richard D. Hayward, University College London and Birkbeck, University of London, UK Glen C. Ulett, Griffith University, Australia*

#### *\*Correspondence:*

*Jason A. Rosenzweig, Department of Biology, Center for Bionanotechnology and Environmental Research, Texas Southern University, 3100 Cleburne Street, Houston, TX 77004, USA e-mail<EMAIL_ADDRESS>Ashok K. Chopra, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070, USA e-mail<EMAIL_ADDRESS>
Like other pathogenic bacteria, *Yersinia* and *Aeromonas* species have been continuously co-evolving with their respective hosts. Although the former is a bonafide human pathogen, the latter has gained notararity as an emerging disease-causing agent. In response to immune cell challenges, bacterial pathogens have developed diverse mechanism(s) enabling their survival, and, at times, dominance over various host immune defense systems. The bacterial type three secretion system (T3SS) is evolutionarily derived from flagellar subunits and serves as a vehicle by which microbes can directly inject/translocate anti-host factors/effector proteins into targeted host immune cells. A large number of Gram-negative bacterial pathogens possess a T3SS empowering them to disrupt host cell signaling, actin cytoskeleton re-arrangements, and even to induce host-cell apoptotic and pyroptotic pathways. All pathogenic yersiniae and most *Aeromonas* species possess a T3SS, but they also possess T2- and T6-secreted toxins/effector proteins. This review will focus on the mechanisms by which the T3SS effectors *Yersinia* outer membrane protein J (YopJ) and an *Aeromonas hydrophila* AexU protein, isolated from the diarrheal isolate SSU, mollify host immune system defenses. Additionally, the mechanisms that are associated with host cell apoptosis/pyroptosis by *Aeromonas* T2SS secreted Act, a cytotoxic enterotoxin, and Hemolysin co-regulated protein (Hcp), an *A. hydrophila* T6SS effector, will also be discussed.

**Keywords: type 2-, -3, and -6 secretion systems, apoptosis, pyroptosis, actin cytoskeleton, effector proteins**

## **INTRODUCTION**

Intricate host-pathogen interactions are constantly evolving as the latter has to combat formidable host immune defenses, resulting in expression/de-repression of genes and molecular mimicry. In some instances, strong immune responses to microbes select for escape mutants with the latter further honing the immune response to that variant pathogen, leading to an ongoing battle, as can be seen typically in human immunodeficiency virus escape mutants (Akahoshi et al., 2012; Yagita et al., 2013) and in *Chlamydia trachomatis* (Nunes et al., 2010). One classic example of this paradigm is programmed host cell death (caused by apoptosis, pyroptosis, and necrosis) which could benefit the host immune system (if clearing an intracellular pathogen) or could be co-opted by the pathogen as a means of eliminating undesirable host cells (e.g., innate immune cells) (Ulett and Adderson, 2006).

Whereas apoptosis is a "self-contained" event that does not stimulate a robust inflammatory response, both pyroptosis and necrosis of host cells release pro-inflammatory cytokines and their cytoplasmic contents into the extra-cellular milieu (Lamkanfi and Dixit, 2010). Apoptosis is a caspase-dependent process that drives embryonic development and is largely characterized by nuclear fragmentation and condensation, blebbing of the plasma membrane, and cell shrinkage. Since all of these physiological consequences occur intracellularly, no host cell cytoplasmic content is released into the extracellular environment, thereby preventing inflammation (Strasser et al., 2000).

Necrosis, by contrast, is a caspase-independent process that results in host cell swelling, disorganized and extensive chromatin hydrolysis, and cytoplasmic leakage (Berghe et al., 2010). Finally, caspase-1-dependent pyroptosis leads to secretion of pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. Caspase-1, interestingly, is not involved in apoptosis and is activated by one of four inflamasomes, which contain a member of the nucleotide-binding oligomerization domain-containing protein (Nod)-like receptor family, during pyroptosis (Lamkanfi and Dixit, 2009). Surprisingly, the pathogenic yersiniae can induce apoptosis, necrosis, and pyroptosis depending on the host-cell type infected (Monack et al., 1997; Ruckdeschel et al., 1997, 1998, 2001; Bergsbaken and Cookson, 2007; Zheng et al., 2012). Similarly, *Aeromonas* species are also able to induce apoptosis (through various caspase activation) (Galindo et al., 2004b, 2006a,b; Martins et al., 2007; Su et al., 2007; Sierra et al., 2007, 2010).

Gram-positive pathogens have also been shown to induce apoptosis in various cell types by disparate mechanisms. Nonsecreted lipotechoic acids (LTAs), well-conserved surface antigens on a wide variety of Gram-positive organisms, are such examples that induce apoptosis by distinct mechanisms in various cell types (Ulett and Adderson, 2006). Beyond LTAs that induce host cell apoptosis, *Bacillus anthracis* employs its lethal factor exotoxin (Park et al., 2002; Popov et al., 2002), *Listeria monocytogenes* utilizes listeriolysin, a cytolysin (Carrero et al., 2004), while the streptococci employ hemolysins (Ring et al., 2002; Liu et al., 2004). Ultimately, Gram-positive pathogens' apoptosis-inducing mechanisms are very diverse; they can typically be either intrinsic (e.g., mitochondrial dysfunction) or extrinsic whereby death domains are activated (Ulett and Adderson, 2006). Of the 27-members that belong to the *Aeromonadaceae* family, *A. hydrophila*, *A. veronii,* and *A. caviae* are frequently isolated as human pathogens, with most infections contracted *via* the fecal-oral route or through wounds (Altwegg et al., 1991; Kirov, 1993; Palu et al., 2006; Edberg et al., 2007). *Aeromonas hydrophila* is also a fish pathogen that can negatively impact the fishing industry. The majority of human *Aeromonas* infections result in self-limiting gastroenteritis or superficial skin infections. However, more threatening systemic infections include bacteremia, cellulitis, peritonitis, hemolyticuremic syndrome, and necrotizing fasciitis (Chopra et al., 1993, 1996; Janda et al., 1994; Merino et al., 1995; Kuhn et al., 1997; Chopra and Houston, 1999; Minnaganti et al., 2000; Brouqui and Raoult, 2001; Galindo et al., 2006a,b; Sha et al., 2013). Alarmingly, it seems as though instances of *Aeromonas*-induced necrotizing fasciitis are on the rise (Huang et al., 2011; Chang et al., 2012; Kao and Kao, 2012; Wu et al., 2012).

Of the 11 known Gram-negative *Yersinia* species, only *Y. enterocolitica*, *Y. pseudotuberculosis*, and *Y. pestis* are human pathogens. Strikingly, while *Y. pseudotuberculosis* and *Y. enetrocolitica* cause self-limiting gastroenteritis (Galindo et al., 2011), *Y. pestis* (transmitted by the bite of an infected flea) causes radically different diseases (bubonic, septicemic, or pneumonic plague) which have resulted in three major human pandemics as well as the great plagues of London in the mid-late 1600s (Inglesby et al., 2000). Currently, the plague-causing bacterium can be treated with various antibiotics (Rosenzweig et al., 2011a), with levofloxacin recently being approved by the Food and Drug Administration against all forms of plague. However, there is no vaccine against this deadly pathogen (Rosenzweig et al., 2011b; Rosenzweig and Chopra, 2012).

As earlier mentioned, representatives of both the *Yersinia* and *Aeromonas* species are capable of causing gastroenteritis following the fecal-oral route of infection, and they similarly possess a type three secretion system (T3SS). The T3SS multiprotein complex/hyperstructure (Norris et al., 2012) is evolutionarily related to the bacterial flagella (Nguyen et al., 2000; Gophna et al., 2003) and enables rapid translocation of effector proteins directly into the targeted host cell cytoplasm, resulting in a number of anti-host consequences. Interestingly, whereas the yersiniae possess well-studied T3SS weaponry, *Aeromonas* species harbor well-defined T3- and T6-secretion systems (derived from phage injection machinery) along with its two identified effector protein substrates, Hemolysin co-regulated proteins (Hcps) and Valine glycine repeat G proteins (VgrGs) (Sierra et al., 2007, 2010; Vilches et al., 2009; Bergh et al., 2013; Sha et al., 2013). Interestingly, hemolysins in *Streptococcus agalactiae*, a Group B streptococci, have also been shown to induce apoptosis in phagocytic cells (Ulett and Adderson, 2006 and references therein). The yersiniae T6SSs have not been as extensively characterized; however, in *Y. psuedotuberculosis*, it is regulated by the transcriptional factor OmpR and appears to play a role in stress responses, quorum sensing, and maintenance of internal pH homeostasis (Zhang et al., 2011, 2013; Gueguen et al., 2013). In *Y. pestis*, the T6SS was found to secrete an Hcp-like autoagglutination factor (Podladchikova et al., 2011). Finally, *Aeromonas* species also employ the general secretory T2SS pathway to export cytotoxic enetrotoxin Act (with hemolytic, cytotoxic, and enterotoxic activities) into the extracellular milieu (Chopra and Houston, 1999). Within the yersiniae, the T3SS injects into the host 7 *Yersinia* outer membrane protein (Yop) effector proteins that have been identified as YopP/J, -H, -E, YopO/YpkA, YopT, YopK, and YopM; these Yops counteract host immune defenses by various mechanisms (Viboud and Bliska, 2005). Upon first encountering a Gram-negative pathogen, like *Y. pestis*, innate immune cells (e.g., macrophages and/or dendritic cells) recognize non-specific, pathogen-associated molecular patterns/microbe-associated molecular patterns (PAMPs/MAMPs), like lipopolysaccharide (LPS), lipoprotein, or flagellin. When PAMPs/MAMPs associate with their recognition receptors, e.g., Toll-like receptors (TLRs), various mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor Kappa B (NF-κB) signaling pathways are activated resulting in the upregulation of IL-12, -18, and tumor necrosis factor alpha (TNF-α) proinflammatory cytokine production (Matsumoto and Young, 2009).

The yersiniae counteract the aforementioned inflammatory response when YopP/J acetylate I kappa B kinase (IKK) and MAPK kinases (MKKs), preventing their phosphorylation and subsequent activation. The disruption in these signaling events results in innate immune cells undergoing apoptosis (Orth, 2002; Mittal et al., 2006; Mukherjee et al., 2006). A more detailed description of *Yersinia* outer membrane protein J (YopJ) mechanisms of mollifying host defenses is discussed in a later section. YopE, -H, -T, and YopO/YpkA all operate to disrupt actin cytoskeleton re-arrangements and phagocytosis, albeit by attacking unique and distinct targets. YopE is a GTPase-activating protein (GAP), while YopT targets Rac-1, RhoA and Cdc-42, and YopH, which is a tyrosine phosphotase, primarily targets focal adhesion complexes. YopO/YpkA, through its kinase activity, also targets Rac-1 and RhoA as well as actin directly. YopM localizes to the target cells' nuclei and disrupts cytokine IL-15 production by targeting ribosomal S6 protein kinase 1 (RSK1) and possibly protein kinase C-like 2 (PRK2) (Matsumoto and Young, 2009 and references therein). Finally, YopK was found to associate with the translocation pore and is believed to modulate inflammation (Brodsky et al., 2010).

As mentioned earlier, many *Aeromonas* species also possess a T3SS. In fact, within the fish pathogen *A. salmonicida*, four T3SSassociated effectors have been identified: AexT, AopP, AopH, and AopO (Braun et al., 2002; Dacanay et al., 2006; Fehr et al., 2006). Our laboratory recently identified an AexT-like protein (a novel T3SS effector, AexU) in a diarrheal isolate SSU of *A. hydrophila* (Sha et al., 2007). While *Aeromonas* outer protein P (AopP) disrupts NF-κB signaling downstream of IKKB, unlike YopJ in the yersinaie, it does not disrupt the MAPK signaling pathway (Fehr et al., 2006). On the contrary, AexT and AexU both possess highly cytotoxic ADP-ribosyltranferase activity for host proteins (Braun et al., 2002; Sha et al., 2007). AopO and AopH remain poorly understood and are homologues of yersiniae YopO/YpkA and YopH, respectively (Sha et al., 2007). Interestingly, we also demonstrated that an *A. hydrophila aopB* deletion mutant, unable to translocate effector Aops into host cells, exhibited greatly reduced virulence in a murine model of infection (Sha et al., 2005).

#### **T3SS EFFECTOR YopJ'S MECHANISMS OF ANTI-HOST ACTIVITY**

The yersiniae T3SS effector YopJ is an acetyltransferase as well as a de-ubiquitinase. Its anti-host activity involves blocking MAPK signaling and NF-κB activation (**Table 1**). This aberrant signaling leads to significantly reduced production of both proinflammatory and anti-apoptotic host cytokines (Monack et al., 1997; Orth et al., 1999, 2000; Mukherjee et al., 2006). Shedding more light on the mechanism of YopJ anti-host activity, a report from Shrestha et al. (2012) identified that YopJ reduced the induction of eukaryotic initiation factor 2 (eIF2) in both yeast and mammalian cells, and that eIF2 signaling was required for YopJ-mediated inhibition of NF-κB activation as well as pro-inflammatory cytokine production.

#### *TLR-2, NF-***κ***B, and Nod2 signaling pathways all targeted by the versatile YopJ*

By using recombinant YopJ (rYopJ), it was determined that TLR-2 in murine macrophages was involved in YopJ-mediated apoptotic signaling by increasing production of caspases 3 and 8, IL-1 receptor associated kinase (IRAK)-4, Fas-associated protein with death domain (FADD), and phosphorylation of IκB and MAPK (Pandey and Sodhi, 2011). Together with this TLR-2 apoptotic signaling, the ability of YopJ to target macrophage eIF2 signaling pathway required for inhibition of NF-κB activation as well as pro-inflammatory cytokine production ultimately leads to host cell apoptosis (Shrestha et al., 2012). In a separate study employing a *Drosophila* model system, transforming growth factor (TGF)-β-activated kinase (TAK1), which is part of the immune NF-κB signaling pathway independent of the TLR-2 signaling, was identified as the YopJ serine/threonine acetylation target (Paquette et al., 2012). Following YopJ acetylation of serine/threonine residues in the active site of *Drosophila* TAK1, its phosphorylation was blocked preventing activation of this kinase. Corroborating *Drosophila* studies, YopJ similarly modified and inhibited TAK1 in mammalian cells (Paquette et al., 2012).

Despite an earlier study demonstrating rYopJ activation of TLR-2 signaling in macrophages (Pandey and Sodhi, 2011), *in vivo* studies employing both *Drosophila* and macrophage models of infection clearly demonstrated that native YopJ indeed activated the NF-κB signaling but not the TLR-2 signaling pathway (Paquette et al., 2012). It was proposed that following acetylation of key serine/threonine residues in the active sites of both RIP (receptor interacting protein 1)-like interacting caspaselike apoptosis regulatory protein kinase (RICK) and TAK1, YopJ prevented the interaction of RICK and Nod2, a NACHT-leucinerich repeats (NLRs) recognition receptors. Further, Nod2 interacted with caspase 1, promoting increased expression/production of IL1-β and dissemination of *Y. psudeotuberculosis* through the gut epithelium (Meinzer et al., 2012). Perhaps this seeming contradiction of YopJ not signaling through TLR-2 during

*MAPK, mitogen-activated protein kinases; NF-*κ*B, nuclear factor Kappa B; TLR-2, toll-like receptor 2; IRAK 4, interleukin 1 receptor associated kinase; FADD, Fasassociated death domain; TAK1, tumor growth factor* β*-activated kinase; RICK, receptor interacting protein 1-like interacting caspase-like apopotosis regulatory protein kinase; Nod2, nucleotide-binding oligomerization domain-containing protein 2; IL 1-*β*, interleukin 1 Beta; T3SS, type three secretion system; Yop, Yersinia outer protein.*

*in vivo* studies while rYopJ was shown to signal through TLR-2 *in vitro* underscores one very important point. Different cell types and/or organisms likely possess disparate/specialized receptors used to recognize threatening pathogens. Viewed in this light, one can envision how within one cell type there could exist several unique receptors that could detect the same (or different products) derived from one pathogen. In the very delicate host-pathogen paradigm, every potential detection mechanism must be employed if the host is to successfully subvert the pathogenic threat. Collectively, YopJ has been observed disrupting TLR-2, Nod2, and the NF-κB signaling pathways, making the T3SS-acetyltransferase a potent weapon for the pathogenic yersiniae.

#### *The yopK "switch" for YopJ activity*

Importantly, YopJ seems to function in concert with another Yop, YopK, which regulates YopJ activity (**Table 1**). Studies have shown that YopK appeared to regulate pyroptosis (via caspase 1) by *Y. pseudotuberculosis* (Brodsky et al., 2010) and YopJ-dependent apoptosis specifically in RAW 264.7 monocytic cells, thereby facilitating bacterial dissemination in a murine model of pneumonic plague (Peters et al., 2013). Despite YopK appearing dispensable for *Y. pseudotuberculosis* to induce caspase-1 mediated pyroptosis (Brodsky et al., 2010), it was required for optimum virulence of *Y. pestis* in a pneumonic murine model of infection (Peters et al., 2013). Further, cell culture *Y. pestis* infection models have also revealed that caspase 1 activation occurs downstream of cell necrosis, is independent of mitochondrial driven apoptosis, but does require cathepsin B activity (Zheng et al., 2012). Taken together, these data demonstrated that, at least in *Y. pestis*, YopJ's ability to induce apoptosis is regulated by a YopK "switch" downstream of cell necrosis.

#### **T3SS EFFECTOR AexU's MECHANISMS OF ANTI-HOST ACTIVITY**

Shortly after a T3SS was identified in both *A. salmonicida* and *A. hydrophila* and implicated in their pathogenesis of both fish and animal/human hosts (Burr et al., 2002; Yu et al., 2004; Sha et al., 2005), we identified a novel T3SS-dependent AexT-like protein, referred to as AexU, in *A. hydrophila* (Sha et al., 2007; Sierra et al., 2007). AexT (**Table 2**), identified in the fish pathogen *A. salmonicida*, is homologous to the *Pseudomonas aeruginosa* ExoT/S and is also a bifunctional effector (Pederson et al., 1999; Sundin et al., 2004). Its amino terminus has YopE-like activity of yersiniae and can depolymerize actin by targeting RhoA, while its carboxy-terminus has highly cytotoxic ADP-ribosyltransferase (ADP-RT) activity for host proteins (Braun et al., 2002).

While the amino terminus of AexU from *A. hydrophila* SSU maintained ∼67% sequence similarity to its AexT counterpart, surprisingly, the AexU carboxy terminus had a unique sequence which did not share similarity with any other known protein in the NCBI database, despite full-length AexU maintaining ADP-RT activity (Sha et al., 2007). Surprisingly, the purified full-length, truncated amino terminus, or truncated carboxy terminus of AexU all exhibited ADP-RT activity; however, the full-length AexU and its amino terminus exhibited higher ADP-RT activity than did the carboxy terminus of AexU alone (Sierra et al., 2007). Since the ADP-RT activity of the *Pseudomonas* homologue (ExoT/S) resides in its carboxy terminus, the aforementioned finding suggested a potentially unique evolution of AexU as an *Aeromonas* T3SS effector.

*RhoA, Rat sarcoma homolog gene family member A; GAP, GTPase activating protein; NF-*κ*B, nuclear factor Kappa B; JNK, c-Jun N-terminal kinase; IL, interleukin; Rac-1, Rac GTPase activating protein 1; MEK1, MAP/ERK kinase 1; ERK1/2, extracellular signal-regulated kinase 1/2; MAPK, mitogen-activated protein kinases.*

Supporting this view, findings from a comprehensive genomic study evaluating an *A. veronii* group collection (derived from both clinical and environmental isolates) revealed that all 20 bacterial isolates possessed a functional T3SS as well as both AexU and AexT effectors. However, whereas AexU had a nucleotide substitution rate of ∼17% in its carboxy terminal region, AexT was much better conserved and demonstrated only a ∼4% substitution rate (Silver and Graf, 2009). Perhaps, the AexU carboxy terminus is evolving independently from its amino terminus allowing for not only the possibility of producing varying alleles but also producing a variety of AexU effectors capable of adapting to changing environments within the host (**Figure 1**)?

#### *Immunogenicity of AexU and its contribution to overall virulence*

In a cell culture infection model, our laboratory found that *A. hydrophila* AexU (**Table 2**) caused actin reorganization and cell rounding, chromatin condensation, and the activation of caspase 3 and 9, all hallmark features of apoptosis (Sierra et al., 2007). Furthermore, we reported that *A. hydrophila* AexU also possessed GAP activity which strongly promoted apoptosis and disrupted actin cytoskeletal rearrangements of the host cells (Sierra et al., 2010). Additionally, it was noted that *A. hydrophila* AexU prevented phosphorylation of c-Jun [a component of the activator protein 1 (AP-1) transcription factor], c-Jun N-terminal kinase (JNK) and IκBα (thereby disrupting their signaling cascades), and inhibited IL-6 and -8 secretion from HeLa cells. Ultimately, AexU inhibited NF-κB and inactivated Rho GTPases in the host cell (Sierra et al., 2010). For reasons unclear at this time, an AexU variant devoid of both GAP and ADP-RT activities, when produced from an *aexU* null mutant of *A. hydrophila*, induced higher mouse mortality and increased pro-inflammatory cytokine production (Sierra et al., 2010). As was noted with YopJ, perhaps, to increase overall bacterial virulence through inflammation, evolutionary deactivation of AexU's activities provides a valuable "switch" for responding to various hosts?

**FIGURE 1 | The bifunctional AexU effector protein.** The two independent activities of AexU are localized on either the NH2- or the COOH-termini. This allows for the possibility of an independently evolving COOH-terminal activity.

In *P. aeruginosa*, it has been shown that the maturation of IL-1β is negatively regulated by ExoS and is dependent on its ADP-RT activity. In other words, ExoS devoid of this enzymatic activity when produced from the bacteria led to increased IL-1β production and pyroptosis of the host cells. However, AexU seemed to behave differently compared to ExoS as the former without the enzymatic activities was unable to alter IL-1β levels (Sierra et al., 2010). *A. veronii* AexU, in a GAP-dependent manner, was able to similarly disrupt actin cytoskeleton by down-regulating Rac-1 in HeLa cells (Abolghait et al., 2011). Additionally, *A. veronii* AexU was found to co-localize with β4-integrin resulting in cytotxicity for the host cells (Abolghait et al., 2011). Collectively, these data strongly demonstrated AexU's versatility as an effector protein by virtue of its ability to disrupt cell signaling, paralyze the host cell, activate caspases (initiating apoptosis), and interact with β4-integren promoting host cell cytotoxicty. When evaluating AexU's contribution to mouse virulence and immunogenicity, we found that an *A. hydrophila aexU* deletion mutant caused significantly less mortality (40% compared to 90–100%) in intraperitoneally-challenged mice than did infection with the isogenic parental strain (Sha et al., 2007). Importantly, rAexU provided protective immunity to mice when subsequently challenged with *A. hydrophila* (Sha et al., 2007). Additionally, the *A. hydrophila aexU* deletion mutant was unable to disseminate within infected mice leaving their lungs, liver, and spleens relatively sterile (Sierra et al., 2010). The aforementioned findings raise the possibility of developing potential subunit and/or live-attenuated vaccine candidates for *A. hydrophila*, which is an emerging human pathogen.

#### *Aeromonas* **T2SS CYTOTOXIC ENTEROTOXIN (ACT) AND T6SS HCP EFFECTOR**

In addition to a functional T3SS, *Aeromonas* species also possess a T6SS (Suarez et al., 2008) as well as secrete a potent enterotoxin Act (**Table 2**) via the T2SS (Chopra and Houston, 1999). In efforts to better understand the host cell response to Act, our laboratory obtained transcriptome profiles of Act-exposed murine RAW 264.7 cells. Not surprisingly, of the 76 differentially expressed genes identified in Act-treated macrophages, many were involved in immune responses, including inflammation (Galindo et al., 2003). Additionally, several apoptosis-related genes were also found to be up-regulated including (but not limited to) Bcl-10 (promotes activation of NF-κB and maturation of pro-caspase 9), BimEL (involved in p38 and JNK-associated apoptosis), and TNF receptor associated factor 1 (TRAF1, which regulates activation of NF-κB and JNK). These transcriptome results with respect to apoptosis-related genes were confirmed by performing real-time PCR as well as functional assays for apoptosis (Galindo et al., 2003).

Since primary host cells may vary in their responses to a stimulant compared to the transformed cell lines, our laboratory evaluated transcriptome profiles of primary murine peritoneal macrophages after treatment with Act. We observed 66% differential gene expression, mirroring our results seen with Act-exposed RAW 264.7 cells (Galindo et al., 2004a,b). However, differential expression of 28 genes unique to primary macrophages was also observed. The pro-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) and Myeloid differentiation primary response 116 (MyD116) genes were upregulated, while interferon consensus binding proteins 8 (IRF-8—involved in immune responses) was downregulated in Act-treated primary cells (Galindo et al., 2004a). When the effect of Act on human HT-29 colonic epithelial cells' transcriptome profiles was evaluated, we noted upregulation of genes involved in immune responses (e.g., IL-8) and apoptosis (e.g., Bcl-2-like genes) as well as phosphorylation of MAPKs (e.g., p38 kinase, extracellular signal-regulated kinase 1/2 [ERK1/2], and JNK) (Galindo et al., 2005), mirroring what was observed earlier in our mouse macrophage studies (Galindo et al., 2003, 2004a,b). Further, through proteomic analysis, we determined that Act increased phosphorylation/activation of cyclic AMPresponse element binding protein (CREB), c-Jun, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) (Galindo et al., 2005).

Realizing that Act induces apoptosis in both cultured and primary macrophages, we elucidated the molecular mechanisms and specifically interrogated the MAPK signaling pathway. We found that, in various cell types, Act exposure resulted in activation of JNK and ERK1/2. Furthermore, Act induced activation/phosphorylation of MAPK upstream factors MKK3/6, MKK4 and MAP/ERK kinase 1 (MEK1) as well as downstream transcription factor c-Jun (Galindo et al., 2004b). With regards to apoptosis, Act induced classical membrane blebbing, increased production of mitochondrial cytochrome *c* and apoptosis-inducing factor, in addition to caspase-3, -8, and -9 activation (Galindo et al., 2004b). When we screened for interactions between Act and both human and yeast proteins (using proArrays), Act was found to bind 9 human proteins (out of ∼1800 proteins screened). Of the 9, synaptosomal-associated protein 23 (SNAP23), galectin-3, and guanylate kinase 1 (GUK-1) were knocked down in murine macrophages and HT-29 epithelial cells (using small inhibitory RNA), with the former two resulting in reduced induction of apoptosis following Act exposure (Galindo et al., 2006a,b). Interestingly, we also observed that DNA adenine methyltransferase (Dam) and Glucose inhibited division protein (GidA) both work to positively influence *act* gene expression and its associated hemolytic activity (Erova et al., 2012). Interestingly, the Gram-positive pathogen, *Staphylococcus aureus*, also secretes a potent, pro-apoptotic enterotoxin, the superantigen enterotoxin B. The aforementioned enterotoxin specifically targets T-cells and activates FAS receptor signaling (Ulett and Adderson, 2006 and references therein).

The *Aeromonas* T6SS has two identified effector protein substrates, Hcps and VgrGs (Sha et al., 2013). *A. hydrophila*'s Hcp (**Table 2**) is a powerful effector substrate and once translocated into the targeted host cell cytoplasm, apoptosis ensues following caspase 3 activation (Suarez et al., 2008). We also demonstrated that Hcp paralyzes macrophages thereby preventing phagocytosis (Suarez et al., 2010). Curiously, multiple copies of Hcp are present in T6SS-possessing bacteria suggesting either redundancy of function and/or dosage-related functional influences (Mougous et al., 2006). The *Aeromonas* gene duplications and various alleles are likely a byproduct of co-evolution occurring in both bacterial pathogens and their respective hosts.

 Hcp-2, located inside the T6SS cluster appeared to function structurally in forming the T6SS apparatus while Hcp-1, located at a distal chromosomal site functioned more as an effector (Sha et al., 2013). More specifically, only Hcp-1 worked to negatively regulate bacterial motility and protease production (both required for optimal virulence) whereas both paralogs were required for optimal virulence and dissemination to peripheral organs in a murine model of infection (Sha et al., 2013). When considering the impressive arsenal available to *A. hydrophila* that includes a T3SS, a T6SS as well as a potent T2SS secreted Act, it becomes less surprising that human infections caused by this emerging pathogen are on the rise.

## **CONCLUSION**

In context of an intricate host-bacterial pathogen co-evolutionary paradigm, at times it become difficult to determine whether the resulting outcomes better benefit the host or the pathogen. For example, following inhibition of NF-κB and MAPK signaling pathways, *Yersinia* species through their generic PAMP/danger signals (e.g., LPS or flagellin), can induce pyroptosis a specialized inflammation-associated apoptosis that involves the activation of caspase 1 (Philip and Brodsky, 2012 and references therein). Inflammation is, in reality, a double-edged sword. If of short duration and localized, it can serve to reduce extent of bacterial infection preventing systemic spread of the pathogen. However, if persistent and/or systemic, inflammation can damage host tissue and potentially promote bacterial spread contributing to bacterial pathogenesis. These two scenarios underscore the intricacies of the host-pathogen interaction as well as reveal how co-evolution can be shaped. The yersiniae T3SS effector, YopJ, is a perfect example of such an ambiguity. Despite its potent immunomodulatory capabilities, YopJ was largely dispensable for virulence in a rat model of bubonic plague (Lemaitre et al., 2006). Further, there has been evolutionary selective pressure against excessive YopJ secretion in order to achieve maximal virulence during plague infections (Zauberman et al., 2006; Brodsky and Medzhitov, 2008). What does this all mean? Does YopJ's powerful ability to induce inflammation benefit the pathogen or the host? Perhaps viewed in this light, the cost to benefit ratio nears "1" making YopJ a "circumstantial virulence factor" depending on the *Yersinia* pathogen in question, the route of infection, the immunodisposition of the host, etc.

The T3SS is a powerful vehicle of effector protein delivery shared by many Gram-negative pathogens. The pathogenic *Aeromonas* species also possess a functional T3SS that delivers 4 effector proteins into targeted host cells. The YopJ homolog, AopP disrupts NF-κB signaling downstream of IKKB but, unlike YopJ in the yersinaie, does not disrupt the MAPK signaling pathway (Fehr et al., 2006). AexU, is an extremely versatile *Aeromonas* T3SS bifunctional effector that possesses both GAP activity (like the yersiniae YopE) as well as ADP-RT activity. Like YopJ, AexU induces apoptosis and targets NF-κB signaling. However, unlike in the yersinaie which lack clearly defined T6SS-virulence factors, *Aeromonas*species possess well-defined T6SS-associated virulence factors and even a T2SS-secreted toxin (Act) creating a much wider arsenal. The T6SS Hcp paralogs sharing limited functional redundancy suggest that co-evolution might have shaped the gene duplication event as well as provide the necessary selection pressure that maintains the multiple copies in the chromosome. Taken together, T3SSs effectors in both the yersiniae and *Aeromonas* species as well as T2- and T6SS effectors in *Aeromonas* species converge on modulating the host immune response to promote bacterial virulence.

#### **ACKNOWLEDGMENTS**

We would like to greatly acknowledge lively discussions and insights provided by Drs. Jian Sha (UTMB, Galveston), Kurt Schesser (University of Miami), and Greg Plano (University of Miami). Work on this review was supported by National Aeronautics and Space Administration (NASA) cooperative agreement NNX08B4A47A (Jason A. Rosenzweig), and NIH/NIAID AI064389 grant awarded to Ashok K. Chopra.

#### **REFERENCES**

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Palu, A. P., Gomes, L. M., Miguel, M. A., Balassiano, I. T., Queiroz, M. L., Freitas-Almeida, A. C., et al. (2006). Antimicrobial resistance in food and clinical *Aeromonas* isolates. *Food Microbiol.* 23, 504–509. doi: 10.1016/j.fm.2005.07.002

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 August 2013; accepted: 14 October 2013; published online: 30 October 2013.*

*Citation: Rosenzweig JA and Chopra AK (2013) Modulation of host immune defenses by Aeromonas and Yersinia species: convergence on toxins secreted by various secretion systems. Front. Cell. Infect. Microbiol. 3:70. doi: 10.3389/fcimb.2013.00070*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*

*Copyright © 2013 Rosenzweig and Chopra. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Role of type I interferons in inflammasome activation, cell death, and disease during microbial infection

## *R. K. Subbarao Malireddi and Thirumala-Devi Kanneganti\**

*Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA*

#### *Edited by:*

*Amal O. Amer, The Ohio State University, USA*

#### *Reviewed by:*

*Mikhail A. Gavrilin, The Ohio State University, USA Dario S. Zamboni, Universidade de São Paulo, Brazil*

#### *\*Correspondence:*

*Thirumala-Devi Kanneganti, Department of Immunology, St. Jude Children's Research Hospital, MS #351, 262 Danny Thomas Place, Suite E7004, Memphis, TN 38105-2794, USA e-mail: thirumala-devi.kanneganti@ StJude.org*

## **INTRODUCTION**

Interferons (IFNs) are broadly classified into three groups, which are denoted as type I, II, and III based on the specific receptor utilization for their signal transduction. The type I IFN family comprises subtypes of IFNα (13 subtypes), IFNβ, IFNω, and IFNε (Pestka et al., 2004; Hertzog and Williams, 2013). All of the type I IFNs bind to a common heterodimeric receptor, called the IFNα*/*β receptor (IFNAR), composed of two chains, IFNAR1 and IFNAR2, that are associated with the tyrosine kinases Tyk2 and Jak1. Activated Tyk2 and Jak1 recruit and phosphorylate several signal transducer and activator of transcription (STAT) family members (**Figure 1**) (Pestka et al., 2004; Platanias, 2005). Activated STAT1 forms a dimer with STAT2, leading to the recruitment of IRF9 and subsequent formation of a heterotrimeric complex called IFN-stimulated gene factor 3 (ISGF3) (**Figure 1**). This complex translocates to the nucleus, where it binds upstream IFN-stimulated response elements (ISRE) and activates the transcription of type I IFN-inducible genes (Pestka et al., 2004; Platanias, 2005). Type I IFNs are classically known for their antiviral immune responses; however, several studies have demonstrated that a wide range of non-viral pathogens can also induce their expression. However, the specific mechanisms and physiological consequences of IFN responses to such pathogens are poorly understood. Various studies have attributed contrasting roles and differential outcomes to type I IFNs in immune responses to diverse microbial pathogens. The ability of IFNs to regulate cell death has been known for a long time and recent studies have started to reveal the specific mechanisms involved.

During the course of evolution, the arms race between bacterial pathogens and host organisms has resulted in the

Interferons (IFNs) were discovered over a half-century ago as antiviral factors. The role of type I IFNs has been studied in the pathogenesis of both acute and chronic microbial infections. Deregulated type I IFN production results in a damaging cascade of cell death, inflammation, and immunological host responses that can lead to tissue injury and disease progression. Here, we summarize the role of type I IFNs in the regulation of cell death and disease during different microbial infections, ranging from viruses and bacteria to fungal pathogens. Understanding the specific mechanisms driving type I IFN-mediated cell death and disease could aid in the development of targeted therapies.

**Keywords: type I IFN, IFNβ, NLR, caspase-1, inflammasome, cell death, Nlrp3**

development of virulence mechanisms by microbes and the reciprocal development of host counter strategies to efficiently defend against them. Cell death has emerged as one of the important aspects of such a race between microbes and the host, exploited by both, while the final outcome is dependent on the specific pathogen and cell types involved. The in depth molecular mechanisms of how type I IFN signaling causes differential outcomes during different microbial infections remains to be established. Here we discuss the role of type I IFNs in regulating cell death and disease in various infection models and highlight its emerging role in inflammasome activation.

## **INTRACELLULAR BACTERIA**

#### *LISTERIA*

*Listeria monocytogenes* (LM), a gram-positive intracellular bacterium that grows rapidly inside host cells, is known to potently induce type I IFN production in mouse (Havell, 1986; O'Riordan et al., 2002) and human (Reimer et al., 2007) macrophages. LM causes life-threatening infections in immunocompromised individuals and may lead to septic abortion in pregnant women (Pamer, 2004). Upon internalization by phagocytes, LM escapes the early phagosome by secreting a hemolytic toxin, Listeriolysin O (LLO) (Portnoy et al., 1988). *Listeria* mutants that do not express the LLO toxin fail to escape the phagosome or to induce IFN-β production. LM-induced type I IFN production is largely independent of TLR signaling and is instead mediated by cytoplasmic RLR- and STING-dependent pathways through the TBK1-IRF3 signaling axis (Ishikawa et al., 2009; Woodward et al., 2010). In addition, *in vivo* studies demonstrated that *Irf* 3−*/*− and *Ifnar*−*/*− mice, which neither make nor respond to type I IFNs, respectively, are highly resistant to *L. monocytogenes* infection (Auerbuch et al., 2004; Carrero et al., 2004; O'Connell et al., 2004). Furthermore, priming with poly(I:C), a

**Abbreviations:** IFN-I, type I interferon; IFNAR, Interferon-α*/*β receptor; ISRE, Interferon stimulated response element; ISGs, Interferon stimulated genes; TLR, TOLL-like receptor; NLR, NOD-like receptor.

**FIGURE 1 | Mechanisms involved in type I IFN production and its receptor signaling.** Several different stimuli including pathogen derived LPS, glycoproteins, RNA and DNA can induce type I IFNs (IFNα and IFNβ) via upstream pattern recognition receptors. TLRs sense the endosomally located PAMPs (pathogen associated molecular patterns) and DAMPs (damage associated molecular patterns) and recruit TRIF (TRL3 and TLR4) or Myd88 (TLR4, 7, 8, and 9), which further transfer the signals downstream to IRF3 and IRF7. RLRs, NLRs, DAI, STING, and related receptors sense nucleic acids in the cytoplasm. The RNA sensors, RIG-I, MDA-5, and LGP2, and DNA sensor STING use the adapter protein MAVS (mitochondrial antiviral signaling protein) to transfer signals to IRFs for type I IFN transcription. RIG-I and MDA-5 also sense ligands generated by RNA polymerase III from cytoplasmic DNA PAMPs. All these pathways utilize the common downstream kinases, TBK1/IKKε for activating Interferon transcription factors. Type I IFNs bind to IFNAR receptor and activate a robust transcriptional pathway through a JAK-STAT signaling pathway. The transcriptional complexes activated by type I IFN signaling bind to specific ISRE/GAS sequences and lead to the expression of several genes important for cell death, cell proliferation and immune responses.

well known type I-inducing agent, results in enhanced death rate in LM-infected WT mice but not in *Ifnar*−*/*− mice (O'Connell et al., 2004), indicating a detrimental role for type I IFNs during *Listeria* infection. Moreover, LM-infected *Ifnar*−*/*− mice were shown to have reduced lymphocyte and splenocyte apoptosis and antibody-dependent neutralization of LLO controlled infection that resulted in reduced pathology (Auerbuch et al., 2004; Carrero et al., 2004; O'Connell et al., 2004). However, Rayamajhi et al. have proposed an alternative mechanism, where type I IFNs-dependent down-regulation of IFNγ receptor results in heightened susceptibility of wild type mice to *Listeria* infection (Rayamajhi et al., 2010).

*Listeria* is known to trigger assembly of multiple types of inflammasomes that include Aim2, Nlrc4, and Nlrp3 for caspase-1 activation in mouse macrophages. During *Listeria* infection, type I IFNs have significant roles in regulation of inflammasome activation and pyroptosis (Henry et al., 2007; Kim et al., 2010; Rathinam et al., 2010; Wu et al., 2010). A few studies have attempted to dissect the relative contributions of each of these inflammasomes and demonstrated that the detection of DNA by AIM2 receptor was indispensable for inflammasome activation and pyroptosis during *Listeria* infection in human PBMCs and mouse macrophages (Kim et al., 2010; Rathinam et al., 2010; Sauer et al., 2010). In addition, Sauer et al. demonstrated that pyroptosis was totally dependent on Aim2, while the Nlrp3 and Nlrc4 inflammasomes were dispensable for this process (Sauer et al., 2010). These reports implicate that type I IFNs play a role in the efficient induction of inflammasome activation and pyroptosis.

## *LEGIONELLA*

*Legionella pneumophila* is an intracellular, gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires' disease. Lipmann et al. reported that *L. pneumophila*-infected mouse macrophages produce IFNβ in a STING- and IRF3-dependent manner (Stetson and Medzhitov, 2006a; Lippmann et al., 2011). By contrast, Monroe et al. have demonstrated that *L. pneumophila* RNA also stimulated a Rig-I*-*dependent IFN response and proposed that *L. pneumophila* RNA, or host RNA, rather than *L. pneumophila* DNA, as the primary ligand that stimulates the host IFN response (Monroe et al., 2009). IFN-αβ inhibits *L. pneumophila* replication in the permissive A/J or CD1 mouse macrophages (Schiavoni et al., 2004). Furthermore, Bastian et al. have reported that *Legionella* is controlled by IFNβ induced in human lung epithelial cells via MAVS and IRF3 (Opitz et al., 2006). Dendritic cells (DCs) and macrophages are capable of restricting *L. pneumophila* growth through NAIP5-dependent caspase-1 activation and cell death. However, DCs were shown to undergo a more rapid apoptosis than macrophages, leading to enhanced restriction of *Legionella* growth (Nogueira et al., 2009). Indeed, eliminating the pro-apoptotic proteins BAX and BAK or over-expressing the anti-apoptotic protein BCL-2 were both found to restore *L. pneumophila* replication in DCs (Nogueira et al., 2009). Furthermore, a sub-population of DCs, plasmacytoid DCs (pDCs) is known to express higher levels of IFNs (Liu, 2005), which can potentially contribute to the higher cell death responses to *Legionella*. How *Legionella*-induced type I IFN promotes cell death is not well understood currently, however, one possible mechanism might be that Type I IFN-dependent up regulation of pro-cell death molecules like BAK and TRAIL can potentiate apoptosis (Fuertes Marraco et al., 2011; Cohen and Prince, 2013).

#### *MYCOBACTERIUM*

*Mycobacterium* spp. are pathogenic intracellular bacteria that cause tuberculosis (TB) and leprosy. Human and mouse myeloid cells secrete type I IFNs in response to mycobacterial infections (Pandey et al., 2009; Berry et al., 2010; Novikov et al., 2011). Blood based profiling has identified type I IFN-induced genes as the most striking characteristic signature of active TB (Berry et al., 2010). In addition, Wu et al. have reported that several TB-induced genes have key transcription factor binding sites for STATs, IRF-1, IRF-7, and OCT-1 (Wu et al., 2012). IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by *M. leprae in vitro* and were preferentially expressed in progressive lepromatous lesions (Teles et al., 2013). Manca et al. have reported that type I IFNs enhance the virulence of *M. tuberculosis* by suppression of Th1 type immune responses. They have also shown that treatment with purified IFN-α*/*β increases lung bacterial loads, resulting in reduced survival in mice (Manca et al., 2001). In addition, another study reported that treatment with exogenous type I IFN results in a striking loss of mycobacteriostatic activity in monocytes and macrophages (Bouchonnet et al., 2002). Furthermore, Mayer-Barber et al. have shown that both IL-1α and IL-1β are critical for host resistance to TB and provided evidence that type I IFNs suppressed IL-1 production (Mayer-Barber et al., 2011). However, early clinical studies suggested that type I IFNs have beneficial effects against pulmonary TB (Giosue et al., 1998; Palmero et al., 1999). Together, these studies indicate that the role of type I IFNs in mycobacterial infections is debatable and requires further research to establish a clear consensus.

### *FRANCISELLA*

*Francisella tularensis* is a gram-negative bacterium and causes tularemia. *F. tularensis* is classified as a Class A agent (having a high potential for use as a bioweapon) by United States regulators, due to its high virulence and ability to spread rapidly. Cytosolic recognition of *Francisella* induces type I IFN production in an IRF3-dependent manner (Henry et al., 2007). *Francisella* induces Aim2 inflammasome-dependent pyroptosis, which further depends on the ability of the bacterium to induce type I IFN production (Henry et al., 2007; Fernandes-Alnemri et al., 2010). Consistent with this, *Francisella*-induced Aim2 inflammasome activation and IL-1β secretion are abrogated in macrophages derived from bone marrow of *Irf3*- and *Ifnar*deficient mice (Fernandes-Alnemri et al., 2010; Jones et al., 2010; Rathinam et al., 2010). Wild-type *Francisella* that can escape into the cytosol induces type I IFN and Aim2 inflammasome activation and host cell death (Mariathasan et al., 2005; Henry et al., 2007; Jones et al., 2010). This observation underscores the importance of cytosolic recognition of bacteria or their components (DNA in the case of *Francisella*) as an important innate immune mechanism to trigger inflammasome activation. Although studies in mice certainly support an important role for Aim2 in immune responses to *Francisella*, its role in human monocytes seems to be less prominent due to its lack of expression and/or induction in response to type I IFNs (Gavrilin and Wewers, 2011).

#### *SALMONELLA*

*Salmonella enterica* serovar Typhimurium (*S. Typhimurium*) is a gram-negative, intracellular pathogen that is quickly cleared by macrophages. This pathogen is a leading cause of acute gastroenteritis worldwide, which is transmitted primarily via the consumption of contaminated food or water. Induction of rapid cell death is a virulence strategy for this pathogen and contributes to dampening host innate immune responses (Lindgren et al., 1996). Robinson et al. have reported that *Salmonella* exploits type I IFN signaling for eliminating macrophages to establish infection (Robinson et al., 2012). Their studies also revealed that type I IFN-induced cell death of the macrophages is mediated by the classical RIP1-RIP3 dependent necroptosis pathway (Robinson et al., 2012). *Salmonella* is detected by NLRP3 and NLRC4 inflammasomes resulting in casapse-1 activation and pyroptosis (Franchi et al., 2006; Miao et al., 2006; Broz et al., 2010). Broz et al. have suggested the existence of an IFN-inducible regulator of caspase-11 that is crucial for activation of non-canonical Nlrp3 inflammasome activation in mutant *Salmonella-* [type 3 secretion system mutant, i.e., *Salmonella* pathogenicity island 1(SPI-1)] infected mouse macrophages (Broz et al., 2012). Their study revealed that *Salmonella* induces expression and activation of caspase-11 through a Tolllike receptor 4 (TLR4)-dependent and TIR-domain containing adaptor-inducing IFN-β (TRIF)-mediated IFNβ signaling pathway. Consistent with this, *Ifnar1*−*/*− or *Irf3*−*/*−, or *Stat-1*−*/*− macrophages infected with mutant *Salmonella* did not process the casapase-11 or activate the non-canonical cell death pathway. Furthermore, *in vivo Casp1*−*/*− mice are more susceptible to Salmonella infection than the *Casp1*−*/*− *Casp11*−*/*− mice (Broz et al., 2012). Thus, these results indicate that caspase-11 mediated cell death results in detrimental effects to the host. Together, it is evident that *Salmonella* exploits type I IFN signaling to rapidly kill the immune cells to cause disease in the host.

## **EXTRACELLULAR BACTERIA**

#### *STAPHYLOCOCCUS*

Staphylococcal infections have recently emerged as a significant problem to human health, due to the emergence of antibiotic resistant strains that cause life-threatening infections, especially in post-influenza exposures (Klevens et al., 2007; Martin et al., 2009; David and Daum, 2010). *S. aureus* infected mouse and human epithelial cells produce type I IFN in a STAT3-dependent manner in response to its virulent protein A (Martin et al., 2009). In mouse myeloid DCs and macrophages, TLR9 and IRF1 have important roles (Schmitz et al., 2007), while the cell wall component lipoteichoic acid (LTA) utilizes the IRF1-STAT1 axis in mouse macrophages to induce type I IFNs (Liljeroos et al., 2008). Absence of IFNAR signaling results in protection against lethal *S. aureus* pneumonia infection compared to wild-type control mice (Martin et al., 2009). Following recognition of *S. aureus* α-hemolysin, mouse macrophages undergo pyroptosis in an Nlrp3 inflammasome-dependent manner (Mariathasan et al., 2006; Craven et al., 2009). In contrast to Martin et al., a recent report demonstrated that IFNα induces phospholipid scramblase 1 (PLSCR1) in human lung epithelial cells as part of an innate protective mechanism to a bacterial pore-forming toxin (Lizak and Yarovinsky, 2012) and another study demonstrated a protective role of CpG DNA (a potent inducer of type I IFN production) in a mouse model of *S. aureus* pneumonia (Roquilly et al., 2010). Furthermore, Kaplan et al. have shown that phagosomal degradation and cytosolic release of intracellular ligands are essential for the induction of IFN-β in mouse and human DCs, which is required for the host defense against *S. aureus* during cutaneous infection in mice (Kaplan et al., 2012). Taken together, these reports suggest that type I IFNs can have both protective and detrimental roles during *S. aureus* infection. The disease outcome is variable and may depend on the immune status of the host, the site of infection and the specific strains causing the infection.

#### *STREPTOCOCCUS*

*S. pneumonia* causes acute lung infections and activates type I IFN expression (Joyce et al., 2009; Parker et al., 2011). DAI (DNAdependent activator of IFN-regulatory factors) dependent recognition of bacterial DNA is proposed to be responsible for inducing type I IFN expression through cytoplasmic DNA sensing pathway involving STING, TBK1 and IRF3-dependent signaling pathways (Parker et al., 2011). Type I IFN treatment enhances protection of mice against *S. pneumoniae* (Weigent et al., 1986). However, prior exposure to influenza A virus leads to increased susceptibility to bacterial infections in a type I IFN-dependent manner (Morens et al., 2008; Shahangian et al., 2009). As opposed to bacterial infection alone, type I IFNs produced during secondary infection with *S. pneumonia* inhibits production of chemokines like CXCL1 and CXCL2 and sensitizes hosts to secondary bacterial pneumonia (Shahangian et al., 2009).

#### *PSEUDOMONAS*

*Pseudomonas aeruginosa* is a causative agent of pneumonia and infection in cystic fibrosis (CF) patients is associated with significant mortality (Zhuo et al., 2008). *P. aeruginosa* induces type I IFN expression predominantly through the TLR4-TRIF-IRF3 axis (Parker et al., 2012). TLR4 signaling was shown to be important for clearance of *P. aeruginosa* from the lungs and preventing sepsis in infected hosts (Faure et al., 2004; Ramphal et al., 2005; Skerrett et al., 2007; Cohen and Prince, 2013). Similarly, *Trif* and *Irf* 3 deficiencies in mice resulted in reduced expression of type I IFN-induced chemokines including CXCL10 (IP-10) and CCL5 (RANTES) and abrogated neutrophil recruitment to the lungs leading to impaired bacterial clearance (Power et al., 2007; Carrigan et al., 2010). These results indicate a protective role for type I IFNs during *P. aeruginosa* infection (Roy et al., 2013). *P. aeruginosa* infection of mouse macrophages activates the Nlrc4 inflammasome and induces pyroptosis in a flagellin independent manner (Sutterwala et al., 2007). However, later studies demonstrated a requirement of cytosolic flagellin for Nlrc4 inflammasome activation (Miao et al., 2008; Arlehamn and Evans, 2011). Bacterial expression of specific adhesins, lipopolysaccharide, and a functional type III secretion system were all shown to be necessary to evoke apoptosis (Sutterwala et al., 2007) and the cytotoxin, ExoU-expressing *P. aeruginosa* strain has been shown to inhibit caspase-1 dependent pyroptosis (Sutterwala et al., 2007).

#### *ANTHRAX*

*Bacillus anthracis* is a gram-positive, aerobic bacterium that causes severe pulmonary, gastrointestinal, and cutaneous infections (Dixon et al., 1999). Production of the lethal toxin (LeTx) by this bacterium causes extensive cell death, tissue damage and systemic disease. LeTx is composed of a protective antigen (PA) and lethal factor (LF). Gold et al. found that endogenous IFNs (type I and II) inhibit the germination of *B. anthracis* spores, but exogenous application enhanced inflammation thereby increasing mortality (Gold et al., 2007). In addition, Walberg et al. showed that recombinant murine IFNβ or type I IFN inducers like poly(I:C) provide marked protection against "Inhalation Anthrax" (Walberg et al., 2008). *B. anthracis* LeTxs activate the Nlrp1b inflammasome and pyroptosis in mice (Boyden and Dietrich, 2006; Bergsbaken et al., 2009). It is recognized that macrophages from inbred mice may or may not be sensitive to *B. anthracis-*induced pyroptosis based on the presence of the Nlrp1b inflammasome responsive *Nlrp1bS/S* or non-responsive *Nlrp1bR/R* alleles (Moayeri et al., 2010; Terra et al., 2010). In this case, pyroptosis of macrophages is believed to counter anthrax infection since IL-1β released during this process helps to generate protective neutrophil responses. It will be essential to study the contribution of type I IFN in pyroptosis induction during *B. anthracis* infection and investigate whether type I IFN can promote apoptosis of neutrophils and initiate other possible detrimental effects.

#### **VIRUSES**

IFNs were originally discovered as antiviral molecules. Viruses are considerably smaller than other microbial pathogens but represent a major threat to human and other animal health. Extensive progress has been made in understanding the mechanisms of type I IFN production in response to viruses (Stetson and Medzhitov, 2006b; Gonzalez-Navajas et al., 2012; MacMicking, 2012). The diverse mechanisms of cellular entry and tropism of viruses are detected by different TLRs that are strategically located in different cellular compartments (**Figure 1**). Endosomally-located PRRs including TLR3, 7, 8, and 9 are known to trigger type I IFNs (**Figure 1**). TLR7 and TLR9 have particularly important roles in plasmacytoid DCs. Once the virus enters the host cytoplasm, several cytoplasmic receptors such as RIG-I-like receptors (RLRs RIG-I, MDA5 and LGP2) and NOD-like receptors (NOD1 and NOD2) monitor the cytoplasm for microbial PAMPs and initiate type I IFN production and associated immune responses (**Figure 1**).

Programmed cell death is a critical host defense against viruses and type I IFNs are known to be involved to this process. Multiple viruses have been discovered to encode proteins that function to subvert host-induced cell death during infection (Bowie and Unterholzner, 2008; Galluzzi et al., 2010; Kaminskyy and Zhivotovsky, 2010; Gregory et al., 2011). Death of the infected cells is detrimental to viral replication and amplification of viral progeny. However, death of the infected cells can also facilitate viral egress and enhance pathogenesis. Therefore, different viruses have evolved complex mechanisms to enhance or inhibit different forms of cell death (Kaminskyy and Zhivotovsky, 2010). Microarray based studies revealed that a large number of genes are regulated by type I IFNs and several of them are involved in cell death (Der et al., 1998; de Veer et al., 2001; Hertzog and Williams, 2013; Rusinova et al., 2013). However, the mechanisms driving cell death that involve proteins encoded by many of these genes are still awaited.

Uncontrolled chronic viral infections can result in sustained expression of type I IFNs with detrimental pathophysiological outcomes. Two recent studies reported the role of type I IFNs in viral persistence during lymphocytic choriomeningitis virus (LCMV) infection (Teijaro et al., 2013; Wilson et al., 2013). Results from these reports show that robust and acute type I IFNs secreted during early in the infection serve to control viral replication and spread by promoting apoptosis of infected cells and enhancing T cell activation. During chronic infection, prolonged expression and exposure to type I IFNs leads to lymphocyte exhaustion, and reduced viral clearance due to the presence of increased immunosuppressive molecules like IL-10 and PD-L1. Loss of circulating pDCs has been documented in chronic viral infections in mice and humans, which correlates with uncontrolled viral loads, reduced T cell counts and onset of opportunistic infections (Swiecki et al., 2011). However, the mechanism of type I IFN-dependent pDC apoptosis is not entirely clear and represents an important subject for future research.

Type I IFNs promote cell death in multiple ways. Balachandran et al. have demonstrated that type I IFN and protein kinase R (PKR) can sensitize cells to apoptosis primarily through the FADD/caspase-8 pathway (Balachandran et al., 2000). In this study, stimulation of mouse cells with IFN-α*/*β resulted in enhanced apoptosis and reduced viral replication. In a follow up study, Ezelle et al. showed that the poxvirus-encoded protein CrmA was able to inhibit both viral infection- and dsRNA-mediated apoptosis (Ezelle et al., 2001). Both HSV and vaccinia virus affect PKR and RNase L-mediated apoptosis pathways that are activated by dsRNA products released during viral replication (Der et al., 1997; Diaz-Guerra et al., 1997; Kibler et al., 1997). Influenza viruses cause severe lung pathology leading to lung failure and mortality. Type I IFNs have been recognized to mediate induction of pro-apoptotic TRAIL leading to excessive cell death and tissue injury (Hogner et al., 2013). Infection or treatment with type I IFN induces pro-apoptotic genes; IFN-stimulated gene 54 (ISG54) or IFN-induced gene with tetratricopeptide repeats 2 (IFIT2) that promotes apoptosis by mitochondrial-associated BCL2 family proteins (Reich, 2013).

#### **FUNGI**

While type I IFNs are widely known as anti-viral factors, which are either protective or detrimental in bacterial infections, their role in fungal infections is poorly defined. Recently, two reports have shown that *Candida* spp. induce IFN-β in mouse bone marrow-derived DCs (BMDCs) and macrophages (Biondo et al., 2008, 2012; Bourgeois et al., 2011). Different forms of fungal glucans and mannans are sensed by a wide range of innate pattern receptors like TLRs, CLRs, dectins, and mannose receptors and initiate MyD88–mediated NF-κB and MAPK pathways or SYK-CARD9 signaling for cytokine induction and cell death (Netea et al., 2008; Brown, 2011). During fungal infections, type I IFNs are also produced by a TLR-independent pathway requiring RNA sensor MAVS and IRF3 (Inglis et al., 2010). In contrast, Del Fresno et al. have recently reported that *Candida albicans* induces type I IFN in DCs through a DECTIN-1, SYK-, and CARD9 dependent pathway that requires IRF5-mediated transcription but not IRF3 or IRF7 (Del Fresno et al., 2013). Jensen et al. have reported that poly(I:C)-induced or exogenously added IFNα and IFNβ treatments of macrophages suppress anti-*Candida* immune responses and cause death of infected mice (Worthington and Hasenclever, 1972; Jensen et al., 1992; Jensen and Balish, 1993). In addition, *Ifnar*−*/*− mice are extremely resistant to otherwise lethal *Candida* and *Histoplasma* infections (Inglis et al., 2010; Majer et al., 2012). These studies demonstrated that type I IFNs induce severe kidney damage by promoting excessive recruitment and activation of inflammatory monocytes and neutrophils. However, other reports suggest that type I IFN can be beneficial as part of the host immune response to *C. albicans* (Biondo et al., 2011; Del Fresno et al., 2013).

#### **ROLE OF INTERFERONS IN INFLAMMASOME ACTIVATION AND PYROPTOSIS**

Type I IFNs are innate immune effector molecules with strong pro-inflammatory activities, and have been shown to contribute to the high mortality rates in septic shock syndromes (Karaghiosoff et al., 2003; Huys et al., 2009). Type I IFNs also contribute to inflammasome-dependent caspase-1 activation leading to pro-inflammatory pyroptotic cell death (**Figure 2**) (Anand et al., 2011; Franchi et al., 2012). There have been multiple different inflammasomes identified that sense a diverse array of microbial- and damage-associated PAMPs.  Inflammasome-dependent casapase-1 activation and pyroptosis are associated with the production of mature IL-1β and IL-18 cytokines, which generates a pro-inflammatory environment in host tissues (**Figure 2**). Inflammasome-dependent pyroptosis shares features of both apoptosis and necrosis and is tightly regulated by distinct signaling pathways.

#### **NLRP3**

NLRP3 is the most widely studied inflammasome and it requires two signals for its assembly into an active complex (Kanneganti, 2010; Anand et al., 2011). The first signal is TLR-dependent expression of NLRP3, while the second is often a damage related factor such as production of reactive oxygen species (ROS) or membrane damage (Anand et al., 2011). Activated TLRs transfer signals through two major adapters that specify the downstream signaling pathways (Takeuchi and Akira, 2010). The first one, MyD88 is required for NF-κB activation downstream of all TLRs except TLR3. The second, TRIF plays a dominant role in TLR3 dependent NF-κB activation and TLR4-mediated IRF signaling (Fitzgerald et al., 2003a,b; Sato et al., 2003; Yamamoto et al., 2003).

Recently, our lab and other groups have shown that the TLR4-TRIF axis regulates caspase-11 expression and noncanonical Nlrp3 inflammasome-mediated host defense against enteropathogens, *Escherichia coli* (EHEC), *Citrobacter rodentium*, and *Salmonella Typhimurium* (Kayagaki et al., 2011; Broz et al., 2012; Gurung et al., 2012; Rathinam et al., 2012). Consistent with this, Sander et al. have reported that the gram-negative bacterium *E. coli* induces IFN-β and activates the Nlrp3 inflammasome in the absence of virulence factors other than microbial mRNA (Sander et al., 2011). In addition, Rathinam et al. demonstrated that the IRF3-type I IFN-IFNAR-STAT-1 signaling pathway is indispensable for caspase-11 expression and activation of the Nlrp3 inflammasome and pyroptosis (Rathinam et al., 2012). Furthermore, they demonstrated that IFNβ treatment significantly increased pro-caspase-11 expression and that once induced, caspase-11 undergoes spontaneous activation. Broz et al. have observed that IFNAR-STAT-1 axis is important for caspase-11 activation, but not for its expression in *Salmonella* infected macrophages (Broz et al., 2012). In a subsequent review, they speculated that type I IFN-dependent expression of a yetunidentified host molecule may trigger caspase-11 activation or that a unknown bacterial signal is required (Broz and Monack, 2013).

Two recent studies have revealed that several gram-negative bacteria, but not gram-positive bacteria, can activate the noncanonical Nlrp3 inflammasome and identified LPS as their common PAMP responsible for the activation of Caspase-11 (Hagar et al., 2013; Kayagaki et al., 2013). Both of these studies have shown that intracellular LPS is sufficient to induce the activation of caspase-11. Furthermore, these studies revealed that a subcomponent of LPS, lipid A is sufficient to activate the casaspe-11 dependent non-canonical Nlrp3 inflammasome. They presented evidence that when transfected, intracellular LPS or lipid A could activate the non-canonical Nlrp3 inflammasome independent of TLR4 or the TLR4-associated lipid A-binding proteins MD1 and MD2 and even the TLR4 downstream signaling molecules TRIF and IFNAR receptor molecules. In contrast, a study by Guarda et al. reported that type I IFNs suppress Nlrp1 and Nlrp3 inflammasomes in a STAT1-dependent manner (Guarda et al., 2011). Further studies are required to clarify the role of type I IFNs and their precise mechanisms in the regulation of inflammasome activation. Of particular importance is that the intracellular sensor of lipid A is yet to be identified.

Together, regardless of the exact mechanisms, type I IFNs have clearly emerged as crucial regulators of inflammasome activation and pyroptosis (**Figure 2**). Physiologically, caspase-11 triggered pyroptosis is required for surveillance against bacteria that enter the cytosol, such as the sifA mutant of *S. Typhimurium*, an sdhA mutant of *Legionella pneumophila* and *Burkholderia* species (Aachoui et al., 2013). However, type I IFNs are known to be exploited by microbial pathogens to induce the death of immune cells and suppress host immune responses. In support of this, caspase-11-mediated cell death is responsible for pathology and detrimental effects *in vivo* (Wang et al., 1998; Kayagaki et al., 2011). For example, Broz et al. showed that wild type *Salmonella* induces caspase-11-mediated cell death in caspase-1-deficient mouse macrophages, and that caspase-11 increases the bacterial virulence and host cell susceptibility to infection (Broz et al., 2012).

#### **RIG-I AND NLRP3**

Viruses are by far the best-known inducers of type I IFNs and have also been recognized to induce activation of distinct inflammasomes, including those comprised of NLRP3, AIM2, and RIG-I. The first evidence of Nlrp3 inflammasome involvement in antiviral responses showed its role in sensing of both viral RNA and its analog poly(I:C) in mouse macrophages (Kanneganti et al., 2006a,b). *In vivo*, Nlrp3 inflammasome activation protected mice from influenza infection (Allen et al., 2009; Thomas et al., 2009). Poeck et al. have reported that Rig-I from mouse macrophages senses cytoplasmic RNA viruses and assembles an inflammasome (Poeck et al., 2010). More recently, RIG-I was reported to induce type I IFN through a MAVS/TRIM25/RNF135 signaling axis following influenza infection, and was shown to have profound effects on NLRP3 inflammasome activation and IL-1β secretion in human lung epithelial cells (Pothlichet et al., 2013). Together, these studies demonstrate that RIG-I can itself assemble an inflammasome and also contributes toward, type I IFN-dependent potentiation of NLRP3 expression (**Figure 2**).

#### **AIM2**

The cytosolic bacteria *Francisella novicida* and *Listeria* both induce killing of myeloid and lymphoid cells in a manner dependent on Type I IFN signaling, an effect which has been shown to be detrimental to the host (Auerbuch et al., 2004; Carrero et al., 2004; O'Connell et al., 2004; Henry et al., 2007). Several published studies have clearly established that both *Listeria* (Henry et al., 2007; Warren et al., 2008; Kim et al., 2010; Tsuchiya et al., 2010; Wu et al., 2010) and *Francisella* (Henry and Monack, 2007; Fernandes-Alnemri et al., 2010; Jones et al., 2010; Rathinam et al., 2010) activate the Aim2 inflammasome and pyroptosis in mouse macrophages. Type I IFNs prime AIM2 expression (Kotredes and Gamero, 2013) and potentiate cytosolic bacterial DNA recognition for inflammasome activation (Fernandes-Alnemri et al., 2010). Although mice studies show that type I IFN dependent caspase-11 expression is important for the activation of the noncanonical Nlrp3 inflammasome (Kayagaki et al., 2011; Gurung et al., 2012; Rathinam et al., 2012), it is not known if it is also required for AIM2 and RIG-I mediated caspase-1 activation or cell death.

#### **CONCLUSIONS**

IFNs were the first cytokines discovered to have immune regulatory capacity. Despite their clinical application in some treatment regimens, we still do not have a complete understanding of the mechanistic effects of IFNs required to further develop treatments that capitalize their full potential therapeutic effects. A vast amount of past research has been focused on the role of IFNs as anti-viral molecules with a limited number of studies for other microbial infections. Recent studies have clearly indicated a dual role for type I IFNs both in infectious and inflammatory diseases. Despite the potential benefits, it is often challenging to manipulate type I IFNs for therapeutic purposes due to their role in regulating the expression and activation of a huge number of downstream genes, often complicating the conclusions. Part of the problem is that types I IFNs exert differential immunomodulation on diverse cell types, environments, and varying physiological conditions. The pathogen- and host-mediated counter regulatory pathways further complicate IFN-induced responses in cell death and disease.

Recently, type I IFNs were recognized as crucial regulators of non-canonical NLRP3 inflammasome activation and pyroptosis. Although the majority of literature indicates a positive role for inflammasomes in anti-microbial host defense, recent reports indicate detrimental effects due to excessive cell death, inflammation, and collateral tissue damage in vital organs (Lupfer and Kanneganti, 2012). A paradox exists, where inflammasomes intended to defend against cytoplasmic invaders by pyroptosis, which is predominantly protective *in vitro*, however in *in vivo*, if exceeds a certain level, can lead to cell and tissue damage and organ failure resulting in negative outcomes. The paradox for type-I IFNs is that their ability to inhibit microbial spread by inducing cell death is counteracted by apoptotic depletion of immune cells and inhibiting anti-microbial immune responses leading to immune suppression. Future research should explore the detailed molecular mechanisms that are responsible for type I IFN-dependent cell death and inflammasomes activation in the context of immunity and immunopathology. These findings may lead to better-targeted therapeutic interventions to treat inflammatory and infectious diseases.

#### **ACKNOWLEDGMENTS**

We apologize to the numerous investigators whose papers could not be cited because of space limitations. This work was supported by National Institutes of Health grants (AR056296, CA163507 and AI101935), and the American Lebanese Syrian Associated Charities (ALSAC) to Thirumala-Devi Kanneganti.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 August 2013; paper pending published: 17 September 2013; accepted: 24 October 2013; published online: 12 November 2013.*

*Citation: Malireddi RKS and Kanneganti T-D (2013) Role of type I interferons in inflammasome activation, cell death, and disease during microbial infection. Front. Cell. Infect. Microbiol. 3:77. doi: 10.3389/fcimb.2013.00077*

*This article was submitted to the journal Frontiers in Cellular and Infection Microbiology.*