Using NLP to investigate consumers' expectations regarding food retail

Introduction

The 2019 Responsible Consumption Index set up by GreenFlex and the Agency for the Environment and Energy Management (ADEME) [1] shows that 57% of French people believe that the economic system needs to be revised. It indicates that 60% of French people are concerned by the state of the planet and believe urgent action is needed. Similarly, 86% would prefer to live tomorrow in a society less focused on consumption. Hence, consumption is no longer viewed as the satisfaction of individual needs but also as an act involving social and environmental concerns.

From this perspective, it is appropriate to examine people's representations regarding the general issue of food and "eating better" to identify the elements that impact their acts of consumption. In particular, it is worth understanding people's views of the role of retailers so as to better understand their expectations and choices, insofar as distrust of large companies is at its highest since only 27% of French people trust them, which amounts to a loss of 31 points since 2004.

The major food retailers, whose role is crucial in the economic activity of the various countries in which they operate, have changed appreciably. At first, they were focused solely upon the profitability of their outlets; gradually, however, they have shown greater consumer awareness through the Efficient Consumer Response (ECR) approach. Some of their recent positions go beyond economic merit to respond to societal pressure. For instance, efforts have been made regarding packaging, food waste, respect for the producers, environmental sustainability, etc.

This study proposes a new vision of consumers that draws attention to the numerous nested representations that create individuals instead of viewing them solely in a consumption context. The concepts of corporate social responsibility (CSR) and corporate social performance (CSP) are used to show how the food distribution sector has already adapted to the new expectations. At the same time, in order to analyze consumers' transformation, the theory of socialization is used to apprehend both normative behaviors and the more critical ones, together with the wordings used to express people's new expectations, namely what seems to indicate a break in their views of food.

In the light of these insights, over 5,950 propositions regarding the future of food consumption in France are examined; they are collected from the consultation platform make.org [2] and analyzed via natural language processing (NLP) methods based upon machine learning and deep learning. These propositions indicate the broad views of the French concerning the issue of food, which involve both the responsibilities of the various industry players and the consumers' specific expectations of the distribution operators. The analysis indicates that, far from being seen as just intermediaries between industry players and consumers, retailers are viewed as having a genuine role to play regarding their product range, and they are expected to take positions revealing their commitment to the sector. In contrast, the issue of digital technology is absent from comments. These findings show that digitization is not an end in itself but rather a set of means to facilitate the setting up of consumers' new expectations.

Corporate social responsibility

Definition and challenges of CSR

The literature reveals the complexity of the notion of CSR. [START_REF] Carroll | A three dimensional conceptual model of corporate social performance[END_REF] lists 20-odd definitions in use and outlines the common general orientation seeking to cover the corporate responsibilities that go beyond the economic activity. These definitions also include work on the stakeholders or sustainable development [START_REF] Freeman | Strategic management: A stakeholder approach[END_REF][START_REF] Parmar | Stakeholder theory: The state of the art[END_REF]. The common thread running through these approaches is to extend the accountability of private businesses to reflect the notion of "public accountability" [START_REF] Preston | Private management and public policy: The principle of public responsibility[END_REF]. Recently, [START_REF] Shea | Microfoundations of corporate social responsibility and irresponsibility[END_REF] have provided a synthesis of the benefits of CSR and insist on the benefits relating to purchase intentions or the reputation and image of companies.

At the same time, a more critical angle is found in work defining CSR as a power relationship showing the capacity of the different role players in a particular system to exert pressure upon or coerce others (Brenann, 2014). CSR is also viewed as an ideology seeking to divert popular attention so as to enhance the image of private enterprise [START_REF] El-Akremi | La responsabilité sociale de l'entreprise sous l'éclairage des critical management studies: Vers un nouveau cadre d'analyse de la relation entreprise-société[END_REF].

Hence, numerous studies have shown both the significance and the advantage for private enterprise of integrating social and environmental concerns into their various activities [START_REF] Porter | Strategy and society. The link between competitive advantage and corporate social responsibility[END_REF]. The notion of shared value reconciles the different postures and brings to light the advantages for a company of committing to a CSR approach if the starting point is a common shared value [START_REF] Porter | The big idea: Creating shared value. How to reinvent capitalism and unleash a wave of innovation and growth[END_REF].

After this brief overview of the CSR concept, it is necessary to investigate the evolution of the distribution sector.

The evolution of the distribution sector and CSR

The emergence of large food retail in the 1960s is characterized by a period of strong growth and mass purchases and marked by the integral self-service system. The competitive climate encourages the various players in the food distribution sector. At that time, their intervention concerns mainly prices, presented as the only competitive advantage.

Rapidly, the increased number of players, competitive pressure, and the economic downturn compel the players to try to stand out and find a new competitive advantage. At the same time, the retailers increase actions and public communication relating to CSR. Hence, their discourse increasingly includes the issue of CSR [START_REF] Utgard | Retail chains' corporate social responsibility communication[END_REF].

All three major phases of development of CSR may be used to examine the evolution of the retailers' discourse [START_REF] Frederick | From CSR 1 to CSR 2: The maturing of business and society thought[END_REF]Wood, 1991):

-CSR 1 (1950)(1951)(1952)(1953)(1954)(1955)(1956)(1957)(1958)(1959)(1960) corresponds to companies' voluntary commitment, sometimes accompanied by a philosophical thrust, to integrate social and environmental dimensions into their operations. -CSR 2 corresponds more to response actions to societal pressure, and the approach becomes more strategic. -CSP combines the first two visions, but it is much more pragmatic and normative since it is oriented toward performance and the measure of it.

In the domain of food distribution, there are few or no CSR 1 actions since, as mentioned earlier, environmental concerns did not originally take societal dimensions into account. In contrast, there were numerous communications in line with CSR 2. These reflect the desire to stand out by taking external societal pressure into account [START_REF] Ackerman | Corporate social responsiveness[END_REF]. However, the corporate discourse was still focused upon the consumers' interests in the market sphere. Hence, there were campaigns related to the development of in-house brands to better serve consumers. There were also campaigns focusing on consumers' well-being in their day-to-day living. However, at that time, only the market sphere was involved. From the beginning of the 2000s, messages go beyond the focus on consumers to include the various industry players, and producers in particular. The performance-oriented discourse becomes more general and incorporates all industry players. There were campaigns illustrating the link with producers or dealing with people's carbon footprint.

CSR is thus a complex concept; it offers overlapping rather than opposing visions in a useful framework to understand consumers' representations. As consumers too are exposed to complex environments, it appears necessary to change our analysis frameworks. Most studies on consumer behaviors appear to be restricted to the market sphere only and to focus upon one segment of consumer behaviors only, whereas consumers are individuals operating in many different spheres.

Development of consumer expectations: The contribution of the theory of socialization

The studies dealing with consumer socialization have usually focused on children and have revealed the set of patterns children learn very early that impact upon their purchasing decisions in adulthood [START_REF] Churchill | Television and interpersonal influences on adolescent consumer learning[END_REF][START_REF] Ward | Consumer socialization: Initial study results[END_REF]. Ward (1974: p.2) defines consumer socialization as "processes by which young people acquire skills, knowledge, and attitudes relevant to their functioning as consumers in the marketplace. The author adds, however, that the core mechanisms are set in place during childhood and adolescence; adult learning continues, however, and structures may develop and change. Industrialized countries are currently experiencing far-reaching changes related to the climate and the environment, which has induced a genuine awareness of environmental issues that may be expected to influence consumers. As indicated in the introduction, the index set up by GreenFlex and ADEME indicates that 60% of French people are concerned by the state of the planet and believe urgent action is needed.

The traditional models of distribution are showing signs of weakening, which may indicate that the consumer socialization processes have not been taken sufficiently into account, as these currently include numerous societal concerns. This notion is in line with Dubet and Martucelli's (1996) argument that socialization involves tension between normative and cultural internalization and critical distancing.

These issues are required to understand the development of consumer behaviors in the large food retail sector. On the one hand, consumers have internalized the integral self-service model and the principle of mass distribution of consumer goods as children and adolescents. Studies focusing on children have shown their early presence in retail outlets and their role as prescribers, then purchasers, even for product categories that do not really concern them [START_REF] Brée | Kids marketing[END_REF]. On the other hand, the same consumers operate in increasingly diverse environments and have virtually unlimited access to the information that may prompt a more critical view.

These observations are in line with [START_REF] Simonson | Absolute value: What really influences customers in the age of (nearly) perfect information[END_REF] who present an alternative view of the effects of marketing upon consumers. The authors propose a new way of conceptualizing consumers' decision making, on the premise that consumers are now much more informed than previously and can make connections and analyses. Hence, they act more rationally and have different expectations of brands and industry players.

Similarly, the work on "modern man" (Dubet and Martucelli, 1996), much discussed in the 1990s, arises from a more modern society in which people are exposed to an increasing number of players and subject to greater stimulation from the environment. People belong to numerous social circles and are confronted to an increasing number of tasks and roles. Consequently, it is essential for companies to take hold of societal concerns, as revealed by work on CSR and consumer socialization studies. It is clear that consumers' views and expectations regarding distribution players have changed.

Research method

Data

To investigate the development of consumers' views, this study is based upon qualitative data relating to the main theme of "eating better". Its purpose is to analyze people's spontaneous discourse so as to identify the various spheres involved and understand their overall representation in people's minds. The study uses data collected on a citizen consultation platform (make.org) and focuses upon the topic of food in general rather than that of distribution in particular. French people were invited to answer the following question: "How can we enable everybody to eat better?" The consultation thus lists the propositions made by the French people who answered the question freely in one sentence; the sole constraint was the start of the sentence (the form "It is necessary..." was imposed) and its length (140 characters). The format made it possible to identify the different notions entangled in consumers' representations and to explore more fully the specific characteristics of expectations of the food retailers.

Data pre-processing

The purpose of this stage is to facilitate the algorithmic processes by decreasing noisy data and optimizing computation time by reducing the vocabulary size upstream. The fist data preprocessing step involves tokenization, namely cutting up the text into units composed of single words and multi-word items in order to facilitate the next pre-processing step. Punctuation, diacritics, and capitals are deleted. Next, the stop-words are removed. For the study, the list of stop words in the natural language toolkit (NLTK) in Python [3] was used. Finally, stemming reduced the complexity of the corpus vocabulary. This step involved removing the prefix and/or suffix of each word to reduce it to its stem, using the Snowball stemming algorithm developed by Martin Porter and implemented in NLTK in Python [3].

Vector representation of the citizens' vocabulary via the Word2Vec algorithm

The automatic processing of a large volume of text data via machine learning algorithms (such as clustering) requires prior transformation of the text into numerical value. Word embedding methods are thus used to represent words as numerical vectors. Among these methods, the Word2Vec algorithm has been developed by Google research teams directed by Mikolov (Mikolov, Chen et al., 2013). This technique uses artificial neural networks; its specificity is to determine the word vectors in such a way that words that are semantically close have a similar vector representation. This is unsupervised learning; hence, this method can be used for any raw text, without any prior manual coding; namely, there is no human coding done beforehand. The Word2Vec is based upon a skip-gram neuronal architecture; the purpose of this deep learning algorithm is to provide a vector representation of each word, taking the context surrounding the word (i.e. the words around this word) into account. To this end, a hidden layer neural network perceptron was defined. It is the hidden layer that defines the space of word representations. The neural network inputs the word and predicts an output word context (see Figure 1).

Using the vector representations of words, a vector representation of the citizens' propositions becomes possible. The vector representation of a proposition is thus the average of the word vectors making up this proposition (Milolov, Suttskever et al., 2013).

Results

These word embedding methods were used to meet the two research goals of the study:

• Determining the main themes arising from the citizen propositions to better understand people's perceptions of the role of large retail outlets in the "eating better" paradigm.

• Determining the words that are semantically close to the notion of "large retail outlets" in the citizens' vocabulary.

Automatic detection of themes

To identify the main themes mentioned in the citizens' propositions, a clustering technique was used on the vector representations of these propositions, using a k-means algorithm. Figure 2 shows the word clouds representing the seven main themes that emerged from this classification.

For each word cloud, the descriptive vocabulary and the vocabulary of action (i.e. verbs) were isolated so as to identify both the lexical field and the forms of action considered by the respondents. Table 1 shows the description of the seven themes and their significance in the respondents' discourse.

Examining all seven themes and the nature of the related actions indicates that the issue of food does involve all stakeholders [START_REF] Freeman | Strategic management: A stakeholder approach[END_REF]. Consumers convey a sense of responsibility when they say they need to eat healthier products (less processing, no additives, organic food, etc.), develop individual farming areas (e.g. vegetable gardens), and cook more often. There are answers such as "It is necessary to eliminate processed and highly processed food products, with too much salt, sugar, etc. and to cook raw foods"; " It is necessary to eat organic and local food, take back control, find the time to cook"; "It is necessary to grow our own vegetables"; "It is necessary to accept to spend more time and money to make responsible food purchases (good shopping channel, fair pricing)"; " It is necessary to cook raw products ourselves instead of buying industrial ready meals". At the same time, respondents show they wish to exert power through their consumption when they write, "It is necessary to penalize or boycott the industry players who show no concern for the environment or consumers' health."

Consumers also believe that the industry players have a role to play regarding the quality of the products they sell by opting for local producers, respecting the seasons, and reducing packaging. There are phrases such as "It is necessary that the industry players rethink their packaging. Example, the yogurts still packed in cardboard"; "It is necessary that the industry players indicate clearly the nutritional values, the harmful or borderline components and in bold"; "It is necessary that the industry players should stop getting their raw materials from intensive farming before preparing their products".

The responsibility of producers and farmers is also mentioned; they are expected to adopt a sustainable production approach that is animal-and environment-friendly. Respondents make numerous propositions such as "It is necessary that in agriculture and farming, people stop doing senseless stuff, for the sake of a better world and respect for animals"; "It is necessary that we stop intensive farming and go for animal well-being and human health"; "It is necessary that we eat dairy products (milk, eggs) for which we know that the animals are well treated and roam freely"; "It is necessary to stop pesticides. Nature works better on its own."

Regarding the public regulatory sphere, expectations cover the fields of education (especially of children) and urban planning (creating areas of urban farming on roofs, in the center of towns). There are propositions such as "It is necessary that food education be prioritized at the level of kindergartens! They need to understand the impacts of the food they consume!"; "It is necessary that people are educated so they understand that their everyday consumption has an impact upon the environment"; "It is necessary to establish vegetable gardens everywhere in the cities, they must be free, and everyone should look after them"; "It is necessary to increase the number of vegetable gardens in town, including hanging on the balconies and roofs of new buildings!"

Other expectations include a stricter regulatory environment for the various industry players. Hence, there are numerous propositions couched in terms of prohibitions, obligations, or restrictions, such as "It is necessary that all industry players be forced to produce products with less fat, sugar, and salt contents"; "It is necessary that industry players be prohibited from using plastics in various forms"; "It is necessary that the benefits of large retailers are monitored so as to ensure that the small producers are well paid"; "It is necessary to stop the use of pesticides in our country and the importation of products containing GMOs and pesticides"; "It is necessary to prohibit definitively palm oil (whether organic or not) in all our food, and colorants and non-natural preservatives"; "It is necessary to make compulsory the display of all the environmental characteristics of products (pesticide use, CO2 impact)".

Overall, the descriptive vocabulary used for the actions to be taken shows consumers' comprehensive and critical views that correspond to the duality found in the theory of socialization. Numerous propositions worded as prohibitions or obligations show consumers' lack of support for industry players, producers, farmers, and retailers. Retailers see their role confined to what they offer consumers. They are expected to promote access to healthy products, in particular by choices centered upon consumer interests rather than their own economic performance. There are propositions such as "It is necessary to choose local organic producers, within relatively short distances, to negotiate reasonable prices for clients"; "It is necessary that they offer us more organic products and the meat of farm-raised animals that live outdoors."; "It is necessary that retailers select and really promote the products of better eating."; "It is necessary that retailers (shops) buy their products from local producers".

Their action is not found exclusively in any category, but retailers appear to play a decisive role because they are related to very many recommendations concerning different themes. Hence, the lexical field of large retail outlets was examined to understand better consumers' expectations of them.

Focus upon large retail outlets and digital technology

First, the words whose vector representations are close to the vector word "distribution" were identified: retailer, supermarket, shop, and large superstore. Next, a T-distributed Stochastic Neighbor Embedding (t-SNE) reduction technique [START_REF] Maaten | Visualizing data using t-SNE[END_REF] helped visualize these results in two dimensions. Figure 3 shows the projection of 3,600 word vectors in this space, to which a focus upon large retail-related vocabulary is added.

Over 6% of all propositions include this vocabulary relating to large retail outlets, and they are found within two themes (see Table 2).

The representation of retailers' responsibility in the future of food is thus shown. Retailers are expected to contribute, through their actions, to promoting both the consumption and the production of healthy, organic, and local products. They should also facilitate access to these products (Table 2). Close examination of these propositions shows that the expectations of retailers concern very different spheres. Generally, expectations crystallize consumers' desire for actions that are not directed solely towards profit making but that include taking into account the societal issues related to consumption and ecology.

Hence, there are expectations regarding the product range on offer. Indirectly, consumers expect retailers to show their power through their offering, which is evidenced in propositions such as "It is necessary that stores propose tasty products, not just looking good"; "It is necessary that supermarkets respect the seasonality of products and accept to propose damaged goods and not throw away"; "It is necessary to promote small farms and pesticide-free farming in short distribution channels in all retail outlets, including the large ones".

There are also many propositions that go beyond the traditional role of retailers and indicate expectations of commitment, which is congruent with studies consequent to CSR orientations that draw attention to the significance of managing simultaneously the social, environmental, and financial concerns [START_REF] Elkington | Cannibals with forks: The triple bottom line of 21st century business[END_REF]. Examples of such propositions include "It is necessary that stores such as Carrefour or Leclerc return part of their profits to the farmers or else they should lower their prices"; "It is necessary to find more organic shops in cities [offering products] at affordable prices and promoting neighborhood shops"; "It is necessary that large retailers put pressure on agribusiness to remove all the harmful additives."

In an omnichannel and multichannel environment, the digital transformation of the distribution sector is often viewed as a growth track. As with distribution, no theme is directly related to this notion. First, the words of the lexical field related to digital technology were identified: Internet, digital, drive, online, site, technology, computer. Next, an exploration of the corpus revealed that only 11 propositions are associated with this topic. This result indicates that in this context, digital technology is simply one way of meeting goals that may involve information and learning, linking people, or marketing. All 11 propositions may be grouped under one of these three themes.

Information-related propositions include the following: "It is necessary that people get information from the Internet for their daily menus and then they can cook"; "It is necessary that the nutriscores are indicated on line for the drive-in, and in fact, ideally everywhere. Online, alternatives to the poor scores must be proposed"; "It is necessary that towns and rural areas are reconnected through the creation of a physical and digital space devoted to leaning about taste and good practices"; "It is necessary to set up training programs about food that are freely available on the Internet. Lots of topics need to be discussed".

The theme of linking people is found in propositions such as "It is necessary to set up an Internet platform to promote the sale of local products from farms or neighborhood shops"; "It is necessary that the web sites of stores that sell fool share simple, quick, and cheap recipes" or "It is necessary that we get closer to each other, foster helping behaviors, for example, if someone does not have a fridge, we help him to find one on a donation site."

Finally, the commercial aspect is also brought to light in propositions such as "It is necessary that in the evening, the unsold products are shown on an Internet site that sells hampers for 5 euros instead of throwing away"; "It is necessary that people can order all fresh products on a price-per-kilo on the sites of online shopping"; "It is necessary to buy directly from the market gardeners, and know about them. So, create a web site of all the regions, particularly near the large cities"; and finally, "It is necessary to create reliable sites that propose lists of food products that can suit everybody, and according to the seasons."

The small number of propositions precludes generalization, but they nevertheless indicate that while digital technology may be a useful tool, it does not feature as a priority for consumers.

Conclusion and research avenues

It appears from the analysis that consumers have considerable knowledge about food and that the various spheres they inhabit are intertwined. Consumers are mindful of the needs for better eating, taking into account societal concerns ranging from consideration of the difficulties of producers and farmers to environmental concerns and animal well-being.

Overall, consumers expect retailers to exert their power at the level of the operational leverage they may possess, in particular regarding the range of products they offer. At the same time, they expect full commitment from the retailers, whom they clearly do not see as mere intermediaries but as true contributors at the heart of the societal challenges societies are currently facing. Furthermore, examining the propositions related to prohibitions, limitations, and injunctions reveals that these are mostly directed at the industry players while the notions of commitment and action are associated rather with the retailers. Hence, the latter's image is part of the numerous expectations formulated.

In contrast, digital technology is not a top level concern in the domain of food. Consumers view digital technology solely as a tool, and their expectations are placed elsewhere. It is true that non-food distribution is more concerned with technology-oriented applications, through for example, the concept of customer experience. Food retail, in contrast, is associated with high societal expectations that retailers need to take into account. The respondent profile of the study is not representative of the majority of consumers. Nevertheless, the development of new forms of distribution such as neighborhood shops, new sales outlets such as the French Associations for the Maintenance of Local Farming (AMAP) or local brands bear witness to profound changes in consumers' expectations.

The study results indicate a much less normative and much more critical consumer behavior that is highly likely to intensify. At a time when retailers struggle to generate growth, this study provides an alternative vision of the possible direction retailers could commit their efforts to renew their offering. However, the sheer magnitude of the expectations and the nature of the responsibilities undertaken should alert retailers to the risk of greenwashing. Numerous players are stepping up to speak out on the issue of food, such as the Groupe Carrefour when introducing its Act for Food program, and it is of vital importance to put in place concrete actions. These actions may use digital technology, but that is not an end in itself.

[1] https://presse.ademe.fr/wp-content/uploads/2019/09/Etude2019_CPVF_FR.pdf

[2] https://about.make.org/about-mieuxmanger

[3] https://www.nltk.org/


Juliana K Ariffin

Kaustav Das Gupta

Ronan Kapetanovic

Abishek Iyer

Robert C Reid

David P Fairlie

Matthew J Sweet
email: m.sweet@imb.uq.edu.au.

Histone Deacetylase Inhibitors Promote Mitochondrial Reactive Oxygen Species Production and Bacterial Clearance by Human Macrophages

Broad-spectrum histone deacetylase inhibitors (HDACi) are used clinically as anticancer agents, and more isoform-selective HDACi have been sought to modulate other conditions, including chronic inflammatory diseases. Mouse studies suggest that HDACi downregulate immune responses and may compromise host defense. However, their effects on human macrophage antimicrobial responses are largely unknown. Here, we show that overnight pretreatment of human macrophages with HDACi prior to challenge with Salmonella enterica serovar Typhimurium or Escherichia coli results in significantly reduced intramacrophage bacterial loads, which likely reflect the fact that this treatment regime impairs phagocytosis. In contrast, cotreatment of human macrophages with HDACi at the time of bacterial challenge did not impair phagocytosis; instead, HDACi cotreatment actually promoted clearance of intracellular S. Typhimurium and E. coli. Mechanistically, treatment of human macrophages with HDACi at the time of bacterial infection enhanced mitochondrial reactive oxygen species generation by these cells. The capacity of HDACi to promote the clearance of intracellular bacteria from human macrophages was abrogated when cells were pretreated with MitoTracker Red CMXRos, which perturbs mitochondrial function. The HDAC6-selective inhibitor tubastatin A promoted bacterial clearance from human macrophages, whereas the class I HDAC inhibitor MS-275, which inhibits HDAC1 to -3, had no effect on intracellular bacterial loads. These data are consistent with HDAC6 and/or related HDACs constraining mitochondrial reactive oxygen species production from human macrophages during bacterial challenge. Our findings suggest that, whereas long-term HDACi treatment regimes may potentially compromise host defense, selective HDAC inhibitors may have applications in treating acute bacterial infections. G ene expression in eukaryotic cells is controlled by the acces- sibility of DNA in the form of chromatin wrapped around histone proteins. Histone acetyltransferases (HATs) catalyze acetylation of lysine residues on core histone tails to allow the initiation of transcription, whereas histone deacetylases (HDACs) reverse this process by catalyzing the removal of acetyl groups. Histone deacetylation results in chromatin compaction and the silencing of gene transcription; however, this is a greatly simplified model of gene regulation [START_REF] Minucci | Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[END_REF]. HDACs are grouped into distinct classes based on sequence homology to yeast proteins. The classical HDACs are subdivided into class I (HDAC1, -2, -3, and -8), class IIa (HDAC4, -5, -7, and -9), class IIb (HDAC6 and -10), and class IV (HDAC11) HDACs. They are dependent on zinc for enzymatic activity and share sequence similarity, and most of these enzymes are inhibited by broad-spectrum HDAC inhibitors (HDACi) such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) [START_REF] Gupta | Towards isozymeselective HDAC inhibitors for interrogating disease[END_REF]. In contrast, nonclassical class III HDACs, or sirtuins, function through a distinct NAD ϩ -dependent mechanism. Although commonly used HDACi such as TSA and SAHA target multiple classical HDACs, isoform-selective HDACi for some individual HDACs have now been described. For example, tubacin and tubastatin A preferentially inhibit HDAC6, while MS-275 is most potent against HDAC1 but also inhibits HDAC2 and HDAC3 [START_REF] Gupta | Towards isozymeselective HDAC inhibitors for interrogating disease[END_REF]. HDACi have been found to be effective as anticancer agents, at least for some cancers such as cutaneous T-cell lymphoma, by promoting the expression of tumor suppressor genes and by inducing cell differentiation, growth arrest, and/or apoptosis [START_REF] Falkenberg | Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders[END_REF][START_REF] Li | Targeting histone deacetylases for cancer therapy: from molecular mechanisms to clinical implications[END_REF][START_REF] Mai | Histone deacetylation in epigenetics: an attractive target for anticancer therapy[END_REF][START_REF] Zhang | Histone deacetylase inhibitors and cell death[END_REF]. Although HDACs are termed histone deacetylases, they also act on numerous nonhistone proteins, such as transcription factors, cell cycle proteins, and protein kinases [START_REF] Choudhary | Lysine acetylation targets protein complexes and co-regulates major cellular functions[END_REF]. Hence, HDACi have many effects beyond the regulation of epigenetic states and gene expression.

Beyond their anticancer applications, HDACi have been investigated as therapeutic agents in models of acute and chronic inflammatory diseases [START_REF] Falkenberg | Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders[END_REF][START_REF] Shakespear | Histone deacetylases as regulators of inflammation and immunity[END_REF][START_REF] Sweet | HDAC inhibitors: modulating leukocyte differentiation, survival, proliferation and inflammation[END_REF][START_REF] Halili | Histone deacetylase inhibitors in inflammatory disease[END_REF]. HDACi are reported to inhibit proinflammatory cytokine secretion via multiple mechanisms, including inhibiting the transcriptional activity of hypoxia-inducible factor 1-alpha (HIF-1␣) [START_REF] Shakespear | Histone deacetylases as regulators of inflammation and immunity[END_REF][START_REF] Shakespear | Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages[END_REF], impairing recruitment of proinflammatory transcription factors to target promoters [START_REF] Bode | Histone deacetylase inhibitors decrease Toll-like receptormediated activation of proinflammatory gene expression by impairing transcription factor recruitment[END_REF], reducing the stability of mRNAs encoding inflammatory cytokines [START_REF] Grabiec | Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay[END_REF], and upregulating the expression and activity of the transcriptional repressor complex, Mi-2/NuRD [START_REF] Roger | Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection[END_REF]. Such studies imply that certain HDACs have proinflammatory functions, and indeed this has been reported for a number of HDACs, for example, HDAC4 [START_REF] Wang | Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production[END_REF] and HDAC7 [START_REF] Shakespear | Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages[END_REF]. As proinflammatory cytokines play important roles in host defense, it is a distinct possibility that HDACi may, in dampening inflammation, also compromise host defense. Indeed, this is a major limitation of many existing anti-inflammatory agents, including anti-tumor necrosis factor alpha (TNF-␣) therapy used to treat patients with rheumatoid arthritis and inflammatory bowel disease [START_REF] Bongartz | Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and metaanalysis of rare harmful effects in randomized controlled trials[END_REF][START_REF] Galloway | Anti-TNF therapy is associated with an increased risk of serious infections in patients with rheumatoid arthritis especially in the first 6 months of treatment: updated results from the British Society for Rheumatology Biologics Register with special emphasis on risks in the elderly[END_REF][START_REF] Keane | Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent[END_REF].

Previous studies examining the effects of HDACi on innate immune responses in mouse macrophages reported that treatment with broad-spectrum HDACi, TSA, and valproic acid reduced expression of phagocytic receptors, NADPH oxidase subunits, and inducible nitric oxide synthase [START_REF] Mombelli | Histone deacetylase inhibitors impair antibacterial defenses of macrophages[END_REF]. Consequently, HDACi impaired the phagocytotic activity of mouse bone marrow-derived macrophages and the production of reactive oxygen species (ROS) and nitric oxide. Furthermore, the ability of mouse macrophages to clear Escherichia coli and Staphylococcus aureus upon challenge was compromised [START_REF] Mombelli | Histone deacetylase inhibitors impair antibacterial defenses of macrophages[END_REF]. A genome-wide microarray analysis of gene expression profiles from murine macrophages and dendritic cells stimulated with Toll-like receptor (TLR) agonists also revealed that TSA treatment impaired up to 60% of TLR2 and TLR4 target genes, including pattern recognition receptors, cytokines, chemokines, costimulatory molecules, transcription regulators, and kinases, while only 16% of genes were upregulated [START_REF] Roger | Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection[END_REF]. However, studies on the impacts of HDACi on host immunity have primarily utilized mouse models or mouse cells, and effects on host defense in humans have not been intensively investigated. Although human and mouse innate immune responses are broadly conserved, some phenotypic differences, divergence in gene repertoires, and differential regulation of orthologous genes have been noted between humans and mice [START_REF] Ariffin | Differences in the repertoire, regulation and function of Toll-like receptors and inflammasome-forming Nod-like receptors between human and mouse[END_REF][START_REF] Mestas | Of mice and not men: differences between mouse and human immunology[END_REF][START_REF] Schroder | Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages[END_REF][START_REF] Shay | Conservation and divergence in the transcriptional programs of the human and mouse immune systems[END_REF]. Here we report that, while long-term treatment of human macrophages with HDACi impairs the phagocytic capacity of human macrophages, acute treatment with HDACi at the time of bacterial infection actually enhances mitochondrial ROS (mi-toROS) production from these cells, which correlates with increased antibacterial responses.

MATERIALS AND METHODS

Materials. TSA purified from Streptomyces sp. (Sigma-Aldrich) was dissolved in 100% ethanol, stored at 4°C, and diluted in tissue culture medium to be used at the indicated concentrations. SAHA was synthesized in-house, dissolved in dimethyl sulfoxide (DMSO), stored at Ϫ20°C, and diluted in tissue culture medium to be used at 10 M. Tubastatin A (Sigma-Aldrich) and MS-275 (synthesized in-house) were dissolved in DMSO to 30 mM, stored at Ϫ20°C, and used at 20 M. MitoTracker Red CMXRos (Life Technologies) was stored at Ϫ20°C and was dissolved in DMSO to 1 mM before further dilution in tissue culture medium for the treatment of cells. Human recombinant gamma interferon (IFN-␥) (R&D Systems) was stored at a concentration of 2.5 g/ml at Ϫ20°C and was diluted in tissue culture medium to a final concentration of 2.5 ng/ml. Cytochalasin D (Life Technologies) was dissolved in DMSO to 2 mM, stored at Ϫ20°C, and used at 10 M. MitoSOX Red mitochondrial superoxide indicator (Life Technologies) was dissolved in DMSO to 5 mM, stored at Ϫ20°C, and used at 5 M. pHrodo Green E. coli BioParticles conjugate (Invitrogen) was dissolved in phosphate-buffered saline (PBS) to 1 mg/ml, sonicated for 5 min, vortexed for 1 min, and then stored at Ϫ20°C.

Ethics statement. Before undertaking the studies described, approval for all experiments using primary human cells was obtained from the Medical Research Ethics Committee of the University of Queensland (approval number 2013001519).

Cell isolation and culture. Human monocyte-derived macrophages (HMDM) were prepared from CD14 ϩ monocytes from Red Cross blood donors. In brief, CD14 ϩ monocytes were differentiated into HMDM by culture with 1 ϫ 10 4 U/ml colony-stimulating factor 1 (CSF-1) for 6 days, as previously described [START_REF] Schroder | Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages[END_REF][START_REF] Irvine | Colonystimulating factor-1 (CSF-1) delivers a proatherogenic signal to human macrophages[END_REF]. On day 6, HMDM were harvested and replated in the presence of CSF-1 for experimentation. THP-1 cells were cultured as previously described [START_REF] Schaale | Strain-and host species-specific inflammasome activation, IL-1␤ release, and cell death in macrophages infected with uropathogenic Escherichia coli[END_REF], differentiated for 48 h with 30 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), and rested for 4 h before experimentation. All cells were cultured in an incubator at 37°C with 5% CO 2 .

Bacterial culture. The Salmonella enterica serovar Typhimurium SLI 344 [START_REF] Hoiseth | Aromatic-dependent Salmonella Typhimurium are non-virulent and effective as live vaccines[END_REF] and E. coli MG1655 [START_REF] Blattner | The complete genome sequence of Escherichia coli K-12[END_REF] strains were stored in LB broth containing 50% sterile glycerol at Ϫ80°C. The appropriate multiplicity of infection (MOI) for infection assays was calculated by spectrophotometry analysis of bacterial cell density from an isolated colony grown overnight in LB broth. Possible effects of HDACi on S. Typhimurium and E. coli growth were assessed by spectrophotometric analyses of bacterial cultures grown at 37°C in complete HMDM medium at the indicated time points.

In vitro infection assays. Bacterial infection assays were conducted with HMDM and PMA-differentiated THP-1 macrophages to assess intracellular bacterial loads as previously described [START_REF] Bokil | Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages[END_REF]. In brief, cells were cultured at 4 ϫ 10 5 cells/well in 1 ml antibiotic-free tissue culture medium at 37°C. Then, they were either primed with HDACi for 18 h prior to bacterial challenge (S. Typhimurium or E. coli; MOI, 100) or were cotreated with HDACi at the time of infection. At 1 h postinfection, cells were washed with a medium containing 200 g/ml gentamicin to exclude extracellular bacteria and then maintained in medium containing 20 g/ml gentamicin. In situations where cells were treated with pharmacological reagents, inhibitors were re-added to the medium containing 20 g/ml gentamicin and were maintained throughout the incubation period. At appropriate time points, the medium was replaced with 0.01% Triton X-100 in PBS, and cells were lysed by pipetting. Diluted lysates were cultured on agar plates at 37°C overnight, and colony counts were used to assess intracellular bacterial loads. In some experiments, supernatants were harvested at the indicated time points to assess cell death through the release of the metabolic enzyme lactate dehydrogenase (LDH) from dying cells into culture supernatants. LDH release was quantified by measuring LDH in culture supernatants compared to total LDH (LDH present in supernatants from untreated cells following cell lysis with 0.1% Triton X-100), as previously described [START_REF] Schaale | Strain-and host species-specific inflammasome activation, IL-1␤ release, and cell death in macrophages infected with uropathogenic Escherichia coli[END_REF].

Quantification of phagocytosis. HMDM and PMA-differentiated THP-1 macrophages were plated at a density of 5 ϫ 10 5 cells/well, and on the following day, cells were treated with 10 M cytochalasin D for 30 min at 37°C to inhibit phagocytosis if required and were then treated with 100 g pHrodo Green E. coli BioParticles conjugate with or without HDACi treatment (either priming or cotreatment). Phagocytosis was assessed at 1.5 h posttreatment with pHrodo Green E. coli BioParticles conjugate as measured by flow cytometric quantification of fluorescence using an FACSCanto II flow cytometer (BD Bioscience). FACs data were analyzed using FlowJo 7.6.5 software (Tree Star).

Quantification of mitochondrial ROS. HMDM were cotreated with HDACi and were infected with S. Typhimurium, as above. After 6 h of infection, cells were washed twice with PBS, 5 M MitoSOX Red mitochondrial superoxide indicator (Life Technologies) was added, and cells were incubated for 10 min at 37°C before mitoROS levels were quantified by flow cytometry.

Immunoblotting. Levels of specific proteins were assessed by Western blotting. Briefly, cells were lysed and homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% NP-40) supplemented with 1ϫ protease inhibitors (Roche Life Science). Cell lysates were resolved on precast 4% to 20% gradient SDS-PAGE gels (Bio-Rad) or on 4% to 12% Bis-Tris NuPAGE gels (Invitrogen) and were separated by electrophoresis and then transferred onto nitrocellulose membranes (Bio-Rad). Membranes were probed using anti-acetyl histone H3 (Cell Signaling), antiacetyl-␣-tubulin (Cell Signaling), or anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (Trevigen) antibodies and were then developed by chemiluminescence (Amersham ECL Plus; GE Healthcare).

Statistics. For data combined from 3 or more independent experiments, statistical analyses were performed using Prism 6 software (Graph-Pad) with error bars indicating the standard error of the mean (SEM). For data sets with 3 or more variables, a one-way analysis of variance (ANOVA) was performed followed by Dunnett's multiple comparison test. Data with confidence values of Ն95% (P Ͻ 0.05) were considered statistically significant.

RESULTS

HDACi reduce intracellular bacterial loads within human macrophages. We first investigated the potential impact of treatment with HDACi on the antimicrobial activity of human macrophages that were infected with either S. Typhimurium or E. coli. HMDM and PMA-differentiated THP-1 macrophages were treated with the broad-spectrum HDACi SAHA 18 h prior to infection (priming) or at the time of infection (cotreatment) in order to determine whether potential effects on antimicrobial responses were time dependent. Cells were also primed with IFN-␥ as a positive control for priming of antimicrobial responses. Priming or cotreating cells with SAHA significantly reduced intracellular bacterial loads of S. Typhimurium and E. coli at 24 h postinfection in HMDM (Fig. 1A andB) and PMA-differentiated THP-1 macrophages (Fig. 1C andD). As expected, IFN-␥ priming also reduced intracellular bacterial loads within HMDM. The SAHA-mediated reduction in intracellular bacterial loads may simply reflect the direct killing of bacteria or the effects on macrophage viability of this compound. To exclude these possibilities, we next assessed bacterial growth in the presence of an even higher concentration of SAHA than that which was used in infection assays as well as the release of LDH in cell supernatants following the infection of human macrophages with S. Typhimurium. SAHA treatment did not affect either S. Typhimurium growth (Fig. 1E) or the release of LDH from S. Typhimurium-infected macrophages that had been primed overnight with SAHA (Fig. 1F). Thus, the SAHA-mediated reduction in intramacrophage bacterial loads was not due to the direct killing of bacteria or to reduced macrophage viability. These data also highlight that human macrophages are resistant to cell death triggered by HDACi.

Priming but not cotreatment of human macrophages with HDACi at the time of infection reduces phagocytosis in human macrophages. Another potential explanation for the HDACi-mediated reduction in intracellular bacteria (Fig. 1A to D) is impaired phagocytic uptake rather than enhanced antimicrobial responses. Since treatment with HDACi was previously shown to impair phagocytosis in mouse macrophages [START_REF] Mombelli | Histone deacetylase inhibitors impair antibacterial defenses of macrophages[END_REF], this was a distinct possibility. We therefore examined the effects of SAHA as well as another broad-spectrum HDACi, TSA, on phagocytosis in human macrophages. TSA (1 M) and SAHA (10 M) induced approximately equal levels of hyperacetylation of histone H3 protein (a hallmark of HDAC inhibition) (Fig. 2A), and thus these concentrations were used for subsequent experiments. To assess phagocytosis, the uptake of pHrodo Green E. coli BioParticles was measured by flow cytometry in HMDM and PMA-differentiated THP-1 macrophages that were primed or cotreated with either TSA or SAHA. The actin polymerization inhibitor cytochalasin D was used as a positive control for inhibition of phagocytosis [START_REF] Mimura | Synergistic effect of colchicine and cytochalasin D on phagocytosis by peritoneal macrophages[END_REF]. Priming, but not cotreatment, with either TSA or SAHA reduced phagocytosis by HMDM (Fig. 2B) and PMA-differentiated THP-1 cells (Fig. 2C) as quantified by fluorescence of phagocytosed pHrodo Green E. coli. In contrast, priming with IFN-␥ had no effect on phagocytosis in these assays. Thus, the effect of IFN-␥ priming in reducing intramacrophage bacterial loads (Fig. 1) is consistent with its well-known capacity to boost macrophage antimicrobial responses. Collectively, the above data (Fig. 1 and2) suggest that cotreatment of human macrophages with HDACi at the time of infection does not impair bacterial uptake but promotes bacterial clearance. To more directly address this, HMDM from matched donors were infected with live E. coli or S. Typhimurium isolates and were cotreated with either TSA or SAHA. Cotreatment of human macrophages with HDACi at the time of infection reduced intracellular bacterial loads at 24 h, whereas these treatments had no effect at 1 h postinfection (Fig. 2D andE). Moreover, a direct comparison of HDACi priming versus cotreatment on bacterial loads at 1 h postinfection in PMA-differentiated THP-1 macrophages also showed that cotreatment with HDACi did not result in a reduction of bacterial loads at this acute time point, whereas priming with HDACi did (Fig. 2F andG). In total, these findings show that overnight priming with HDACi impairs macrophage phagocytosis resulting in reduced intracellular bacterial loads, whereas cotreatment with HDACi does not impair phagocytosis but rather promotes bacterial clearance by human macrophages. We therefore focused subsequent efforts on understanding how cotreatment of human macrophages with HDACi promotes antimicrobial responses. These studies concentrated on the professional intramacrophage pathogen S. Typhimurium, given the clinical significance of devising strategies to clear intracellular reservoirs of this pathogen.

HDACi promote mitochondrial ROS release from human macrophages. Previous studies reported that treatment of breast cancer cells and human monocyte lymphoma U937 cells with various HDACi, including TSA, resulted in a loss of mitochondrial membrane potential and enhanced mitoROS production [START_REF] Rehman | Spiruchostatin A and B, novel histone deacetylase inhibitors, induce apoptosis through reactive oxygen speciesmitochondria pathway in human lymphoma U937 cells[END_REF][START_REF] Sun | Trichostatin A targets the mitochondrial respiratory chain, increasing mitochondrial reactive oxygen species production to trigger apoptosis in human breast cancer cells[END_REF]. In addition, mitoROS was previously shown to play an important role in the clearance of S. Typhimurium, at least in mouse macrophages [START_REF] West | TLR signalling augments macrophage bactericidal activity through mitochondrial ROS[END_REF]. Thus, we examined whether HDACi enhanced mi-toROS production by HMDM upon bacterial challenge with S. Typhimurium. Generation of mitoROS in HMDM cotreated with TSA or SAHA during infection with S. Typhimurium was quantified by flow cytometric detection of MitoSOX Red fluorescence. S. Typhimurium infection, which triggers multiple TLRs, resulted in a modest increase in mitoROS production from HMDM at 6 h postinfection (Fig. 3), which is in accordance with a previous report that TLR activation triggers mitoROS production in mouse macrophages at this time point [START_REF] West | TLR signalling augments macrophage bactericidal activity through mitochondrial ROS[END_REF]. Whereas HDACi alone did not promote mitoROS production from these cells, TSA and SAHA significantly enhanced S. Typhimurium-induced mitoROS production. Thus, cotreatment of human macrophages with HDACi during bacterial infection with S. Typhimurium increases mitoROS levels. To determine whether enhanced mitoROS production was likely to be causally related to HDACi-mediated bacterial clearance, we used a pharmacological approach. The mitochondrial stain MitoTracker Red CMXRos is reported to bind irreversibly to the polarized mitochondrial membrane [START_REF] Kroemer | Mitochondrial control of apoptosis[END_REF][START_REF] Marchetti | Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function[END_REF] and is thus predicted to impair the generation of mitoROS. We found that CMXRos blocked HDACi-mediated bacterial clearance (Fig. 4A andB) without having pronounced effects on macrophage viability (Fig. 4C). Although CMXRos-mediated inhibition of mitoROS production was not assessed because this stain interferes with this assay due to spectral overlap, these data implicate enhanced mitoROS production as a likely mechanism accounting for HDACi-mediated clearance of intracellular bacteria.

A specific inhibitor of HDAC6 reduces S. Typhimurium survival in human macrophages. A recent study showed that the HDAC6 inhibitor tubastatin A improved survival and reduced bacterial loads in the blood in a mouse cecal ligation and puncture model of sepsis, whereas this effect was not observed with the class I HDACi MS-275 [START_REF] Li | Inhibition of histone deacetylase 6 improves long-term survival in a lethal septic model[END_REF]. To determine whether HDACi-mediated antimicrobial responses in human macrophages are likely linked to HDAC6 inhibition, we treated HMDM and PMA-differentiated THP-1 macrophages with the HDAC6-selective inhibitor tubastatin A, and for comparison, we also used MS-275, which inhibits the class I HDACs, HDAC1 to -3 (2). As expected, treatment of HMDM with TSA (1 M) or tubastatin A (20 M), but not with MS-275 (20 M), resulted in hyperacetylation of ␣-tubulin (Fig. 5A), which is a hallmark of HDAC6 inhibition [START_REF] Zhao | Histone deacetylase 6 (HDAC6) is an independent deacetylase for alpha-tubulin[END_REF]. Cotreatment with tubastatin A significantly reduced intracellular bacterial loads of S. Typhimurium in HMDM (Fig. 5B) and PMAdifferentiated THP-1 macrophages (Fig. 5D), with 20 M tubastatin A having similar efficacy to 1 M TSA in THP-1 cells. In contrast, cotreatment with MS-275 had no effect on intracellular bacterial loads. Neither tubastatin A nor MS-275 affected cell viability of S. Typhimurium-infected macrophages as assessed by the release of LDH (Fig. 5C andE). These data therefore suggest that HDAC6, but not HDAC1, HDAC2, or HDAC3, constrains infection-induced mitoROS production and that selective inhibition of HDAC6 may promote mitoROS and boost clearance of intracellular bacteria by human macrophages. However, the fact that tubastatin A had effects similar to those of TSA on tubulin hyperacetylation (Fig. 5A), and yet was less effective than TSA in promoting bacterial clearance from HMDM (Fig. 5B), suggests that multiple HDACs may be involved in constraining human macrophage antimicrobial responses.

DISCUSSION

HDACi are known to exert direct antimicrobial effects against some pathogens, such as Plasmodium species (37), and to reactivate latent HIV reservoirs to enable viral clearance [START_REF] Wightman | HDAC inhibitors in HIV[END_REF][START_REF] Margolis | Histone deacetylase inhibitors and HIV latency[END_REF]. As such, HDACi are being pursued as potential therapeutic agents in these infectious disease contexts. Although some mouse studies have suggested that HDACi may compromise host defense against bacterial and fungal pathogens [START_REF] Roger | Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection[END_REF][START_REF] Mombelli | Histone deacetylase inhibitors impair antibacterial defenses of macrophages[END_REF], very few studies have examined the regulation of human macrophage antimicrobial responses by HDACi. Given some differences in innate immune pathways between human and mouse, particularly in relation to host defense [START_REF] Ariffin | Differences in the repertoire, regulation and function of Toll-like receptors and inflammasome-forming Nod-like receptors between human and mouse[END_REF][START_REF] Schroder | Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages[END_REF], we examined the effects of HDACi on human macrophage responses to bacterial challenge in this study.

Cotreatment of primary human macrophages or PMA-differentiated THP-1 macrophages with HDACi reduced intracellular loads of E. coli and S. Typhimurium at 24 h postinfection (Fig. 1A to D) without affecting macrophage viability (Fig. 5C andE), macrophage phagocytic activity (Fig. 2B to G), or bacterial growth (Fig. 1E). Our findings are thus consistent with HDACs constraining human macrophage host defense responses upon bacterial challenge, such that HDACi promote the clearance of intracellular bacteria. Similar conclusions were recently reached using Anaplasma phagocytophilum [START_REF] Garcia-Garcia | Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum[END_REF] and Mycobacterium infection models in THP-1 cells [START_REF] Wang | Mycobacteria inhibition of IFN-gamma induced HLA-DR gene expression by upregulating histone deacetylation at the promoter region in human THP-1 monocytic cells[END_REF]. In our studies, we found that cotreatment with broad-spectrum HDACi enhances S. Typhimurium-inducible mitoROS levels in HMDM (Fig. 3), while treatment of these cells with MitoTracker Red CMXRos, which is predicted to inhibit mitoROS production, abrogates the ability of HDACi to promote bacterial clearance (Fig. 4). Enhanced antimicrobial responses were also observed in human macrophages upon cotreatment with the HDAC6-selective inhibitor, tubastatin A, but not with the HDAC1-selective inhibitor MS-275, also known as Entinostat (Fig. 5). Collectively, these data are consistent with inhibition of HDAC6 and/or related HDACs, enhancing human macrophage antimicrobial responses likely via enhanced mitoROS production.

Previous studies with mouse macrophages have shown that mitochondria are recruited to phagosomes downstream of TLR1, TLR2, and TLR4 activation, leading to the release of mitoROS and to the enhanced killing of S. Typhimurium [START_REF] West | TLR signalling augments macrophage bactericidal activity through mitochondrial ROS[END_REF]. Inhibition of HDAC activity has also been reported to increase the generation of mitoROS in other cellular contexts. For example, treatment of the human breast cancer cell lines MDA-MB-231 and MCF-7 with TSA led to cell death, and this was dependent on mitoROS induction through impairment of the mitochondrial respiratory chain [START_REF] Sun | Trichostatin A targets the mitochondrial respiratory chain, increasing mitochondrial reactive oxygen species production to trigger apoptosis in human breast cancer cells[END_REF]. The HDACi spiruchostatins A and B were also shown to stimulate the production of mitoROS, contributing to the apoptosis of human monocytic lymphoma U937 cells [START_REF] Rehman | Spiruchostatin A and B, novel histone deacetylase inhibitors, induce apoptosis through reactive oxygen speciesmitochondria pathway in human lymphoma U937 cells[END_REF]. Although we found that HDACi boosted S. Typhimurium-inducible mi-toROS generation (Fig. 3), these agents did not enhance cell death in human macrophages challenged with this pathogen (Fig. 1F and5C andE). Thus, human macrophages appear to be somewhat resistant to mitoROS-initiated cell death. However, our finding that treatment with the MitoTracker Red CMXRos stain blocked the ability of broad-spectrum HDACi to reduce bacterial loads within HMDM (Fig. 4) is consistent with a causal role of HDACiinduced mitoROS in the clearance of intracellular bacteria. Due to its cationic lipophilic structure, this stain passively distributes into the mitochondrial matrix to cause monovalent substitution of thiols, preventing changes in the mitochondrial membrane potential [START_REF] Kroemer | Mitochondrial control of apoptosis[END_REF][START_REF] Marchetti | Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function[END_REF], which is linked to the generation of mitoROS (42). However, further mechanistic studies are required to definitively link the effects of HDACi on mitoROS production to bacterial clearance. Enhanced antimicrobial activity was also observed upon cotreatment with the selective HDAC6 inhibitor tubastatin A but not with MS-275 (Fig. 5B andD), which inhibits HDAC1 to -3. This finding is in keeping with a previous study that showed that an HDAC6 inhibitor improved survival and reduced bacteremia in a murine sepsis model [START_REF] Li | Inhibition of histone deacetylase 6 improves long-term survival in a lethal septic model[END_REF]. Moreover, previous studies have linked HDAC6 to the control of mitochondrial function [START_REF] Chen | Identification of novel modulators of mitochondrial function by a genome-wide RNAi screen in Drosophila melanogaster[END_REF][START_REF] Kamemura | Depression of mitochondrial metabolism by downregulation of cytoplasmic deacetylase, HDAC6[END_REF], and a very recent study reported that HDAC6 inhibition disrupted mitochondrial membrane potential and enhanced ROS production in melanoma cells [START_REF] Bai | Down-regulation of deacetylase HDAC6 inhibits the melanoma cell line A375.S2 growth through ROS-dependent mitochondrial pathway[END_REF]. Collectively, these studies suggest that HDAC6 may have a regulatory role in controlling mi-toROS production in infected macrophages such that inhibition of HDAC6 increases mitoROS concentrations to facilitate bacterial clearance. In contrast, on the basis of inhibitor studies (Fig. 5B andD), HDAC1 to -3 (class I HDACs) are unlikely to exert such effects.

Prior studies have reported that priming of mouse bone marrow-derived macrophages with TSA or another broad-spectrum HDACi, valproic acid, reduced the uptake of E. coli and Staphylococcus aureus and impaired the expression of phagocytic and opsonophagocytic receptors in these cells [START_REF] Mombelli | Histone deacetylase inhibitors impair antibacterial defenses of macrophages[END_REF]. Further, the authors also showed that priming with HDACi reduced cellular ROS production at 30 min post bacterial challenge as well as inducible nitric oxide production, leading to impaired bacterial killing by mouse macrophages. Similarly, mouse macrophages and dendritic cells pretreated with SAHA, valproic acid, or TSA had impaired antimicrobial responses to E. coli, S. aureus, and Candida albicans, and in vivo pretreatment with HDACi increased susceptibility to bacterial and fungal infections in mouse models [START_REF] Roger | Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection[END_REF]. These effects were linked to the downregulation of host defense genes such as pattern recognition receptors and costimulatory molecules as well as to the repression of macrophage cytokine production through the Mi-2/NuRD complex [START_REF] Roger | Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection[END_REF]. We similarly found that overnight pretreatment of human macrophages with broad-spectrum HDACi impaired phagocytic responses (Fig. 2B, C, F, and G); however, we did not investigate whether HDACi priming suppressed specific host defense genes in these cells. Overall, these findings may raise some concerns about potential applications for broad-spectrum HDACi, particularly for longterm treatment regimes. However, clinical evidence linking HDACi to infectious disease susceptibility is rather limited so far. Indeed, systemic-onset juvenile idiopathic arthritis patients treated for 12 weeks with the HDACi givinostat concomitantly with glucocorticoids were not reported to display enhanced susceptibility to infections [START_REF] Vojinovic | Safety and efficacy of an oral histone deacetylase inhibitor in systemic-onset juvenile idiopathic arthritis[END_REF]. Moreover, it is quite possible that the anti-inflammatory effects of HDACi may actually reduce the immunopathology that is associated with many infectious diseases.

Nonetheless, concerns remain about the potential for broadspectrum HDACi to increase susceptibility to infectious diseases by reducing phagocytosis and/or inflammation-mediated immune cell recruitment. This highlights the need for studies with selective HDACi, which may offer new opportunities to promote antibacterial responses without compromising host defense. Indeed, a very recent study showed that the HDAC6-selective inhibitor tubastatin A promotes bacterial clearance, reduces inflammatory cytokine production, and improves survival in a mouse cecal ligation puncture model of sepsis [START_REF] Li | Inhibition of histone deacetylase 6 improves long-term survival in a lethal septic model[END_REF]. This exciting finding raises the possibility that pharmacological agents targeting HDAC6 may have dual activities as anti-infective agents in promoting innate immune-mediated bacterial clearance and in reducing immunopathology caused by excessive inflammation. In this regard, bacterial and viral infections are well known to exacerbate inflammation and increase disease severity in various chronic conditions, particularly respiratory diseases [START_REF] Starkey | Murine models of infectious exacerbations of airway inflammation[END_REF]. Moreover, in mouse models, allergic airway disease is associated with impaired host defense responses against bacterial pathogens such as Haemophilus influ-enzae [START_REF] Essilfie | Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma[END_REF]. The potential to reduce inflammation and boost host defense with HDACi may thus be particularly attractive in the context of new therapies for inflammatory diseases of the airways. However, the fact that loss of HDAC2 expression and function has been associated with excessive inflammation and corticosteroid resistance in chronic lung diseases (49) again highlights the need for studies with selective HDACi in this context. Future studies assessing the efficacy of selective inhibitors of HDAC6, as well as other HDACs, in models of infection-mediated exacerbation of inflammatory diseases are thus clearly warranted. More specifically, short-term HDACi treatment may be predicted to reduce infection-induced exacerbations of inflammatory diseases.

In summary, the findings herein reveal that HDACs constrain infection-induced mitoROS production from human macrophages and that inhibition of these enzymes provides a means of enhancing macrophage antimicrobial responses. HDAC-selective inhibitors may provide new prospects as anti-infective agents, and since broad-spectrum HDACi are already approved for clinical use in cancer, there is clear potential for rapid translation of other HDACi into the clinic.

FIG 1

 1 FIG 1 Priming or cotreatment of primary human macrophages with SAHA reduces intracellular bacterial loads of E. coli and S. Typhimurium. Human macrophages were primed with either 10 M SAHA or 5 ng/ml IFN-␥ for 18 h and were then infected with S. Typhimurium or E. coli at an MOI of 100. Alternatively, cells were cotreated with 10 M SAHA at the time of infection. (A to D) Intracellular survival of S. Typhimurium (A and C) and E. coli (B and D) was assessed at 24 h postinfection in HMDM (A and B) and PMA-differentiated THP-1 macrophages (C and D). Data (mean ϩ SEM) are combined from 3 or 4 independent experiments and are presented as fold change relative to untreated controls. ***, P value of Ͻ0.001; ****, P value of Ͻ0.0001 by one-way ANOVA followed by Dunnett's multiple comparison test. p indicates overnight priming with HDACi, and co indicates cotreatment with HDACi at the time of infection. (E) S. Typhimurium was cultured with or without 30 M SAHA for 7 h. Data (mean ϩ SEM) are combined from 3 independent experiments. (F) Cell death was quantified by assessing LDH release into culture supernatants from HMDM primed with 10 M SAHA or 5 ng/ml IFN-␥ for 18 h prior to infection with S. Typhimurium for 24 h. Data (mean ϩ SEM) are combined from 4 independent experiments.

FIG 2

 2 FIG 2 Priming, but not cotreatment, of primary human macrophages with HDAC inhibitors impairs phagocytosis of E. coli. (A) HMDM were treated with 1 M TSA or 10 M SAHA for 6 h, after which cell lysates were prepared and histone H3 hyperacetylation was assessed by Western blotting. Data show immunoblots from one experiment and are representative of 3 independent experiments. Phagocytic capacity of HMDM (B) and PMA-differentiated THP-1 cells (C) after priming or cotreatment with 1 M TSA or 10 M SAHA was measured by uptake of fluorescent pHrodo Green E. coli BioParticles. For cytochalasin D treatment (10 M), cells were treated 30 min prior to treatment with E. coli BioParticles. Intracellular bacterial loads of E. coli (D) and S. Typhimurium (E) in HMDM derived from the same donors as in the phagocytosis assay at 1 and 24 h postinfection are shown after cotreatment with 1 M TSA or 10 M SAHA. Bacterial loads of E. coli (F) and S. Typhimurium (G) in PMA-differentiated THP-1 macrophages at 1 h postinfection, after either cotreatment or overnight priming with 1 M TSA or 10 M SAHA, are shown. Data (mean ϩ SEM) (B to G) are combined from 3 or 4 independent experiments and are presented as fold change relative to untreated controls. **, P value of Ͻ0.01; ***, P value of Ͻ0.001; ****, P value of Ͻ0.0001 by one-way ANOVA followed by Dunnett's multiple comparison test. p indicates overnight priming with HDACi, and co indicates cotreatment with HDACi at the time of infection.

FIG 3 FIG 4

 34 FIG 3 Cotreatment of HMDM with either TSA or SAHA at the time of infection with S. Typhimurium enhances mitochondrial ROS generation. HMDM were cotreated with 1 M TSA or 10 M SAHA during infection with S. Typhimurium at an MOI of 100. At 6 h postinfection, MitoSOX Red was added to cells, and mitochondrial ROS generation (median fluorescence intensity) was measured by flow cytometry. Data (mean ϩ SEM, combined from 3 independent experiments) represent fold change relative to untreated cells stained with MitoSOX Red. *, P value of Ͻ0.05; **, P value of Ͻ0.01 by oneway ANOVA followed by Dunnett's multiple comparison test.

FIG 5

 5 FIG 5 Cotreatment of human macrophages with tubastatin A at the time of infection with S. Typhimurium enhances bacterial clearance. (A) ␣-Tubulin hyperacetylation was assessed in HMDM treated with 1 M TSA, 20 M tubastatin A, or 20 M MS-275 for 6 h. Data show immunoblots from one experiment and are representative of 3 independent experiments. Intracellular bacterial loads of S. Typhimurium in HMDM (B) and PMA-differentiated THP-1 (D) were determined in cells cotreated with either 1 M TSA, 1 to 20 M tubastatin A, or 1 to 20 M MS-275 during infection (MOI, 100). Intracellular survival was assessed at 24 h postinfection. Data (mean ϩ SEM) are combined from 4 independent experiments. Cell death in HMDM (C) and PMA-differentiated THP-1 (E) was assessed by quantifying LDH release from 24-h culture supernatants. Data (mean ϩ SEM) are combined from 3 independent experiments.

aac.asm.org March 2016 Volume 60 Number 3 Antimicrobial Agents and Chemotherapy

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ACKNOWLEDGMENTS

We thank the Australian Red Cross Blood Service for providing buffy coats used for the isolation of primary human monocytes, as well as Daniel Hohenhaus for editorial assistance.

FUNDING INFORMATION

The National Health and Medical Research Council of Australia (NHMRC) provided funding under grants APP1047921 and APP1030169. M.J.S. is supported by an NHMRC Senior Research Fellowship (APP1107914). D.P.F. is supported by an NHMRC Senior Principal Research Fellowship (1027369). A.I. is supported by a University of Queensland Postdoctoral Fellowship and a University of Queensland Early Career Grant. R.K. is supported by an Australian Research Council Discovery Early Career Researcher Award (DE1310470). We thank the Australian Research Council for a Centre of Excellence in Advanced Molecular Imaging grant (CE140100011). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.