**1. Introduction**

Brucellosis is a zoonotic bacterial disease that exhibits pathogenesis consistent with inflammation. Transmitted through *Brucella* spp. primarily from agricultural animals to humans in unpasteurized dairy products, brucellosis symptoms in humans often include inflammatory or influenza-like characteristics such as arthritis, undulant fevers, and neurological manifestations [1,2]. Because there is currently no human vaccine for brucellosis and e ffective antibiotic regiments for the disease require long treatment durations, these symptoms often persist throughout the infected individual's lifetime due to the well-adapted ability of *Brucella* to evade immune recognition [3]. Unlike the classical lipopolysaccharide (LPS) layer of Gram-negative bacteria such as *Escherichia coli* that contain a glucosamine backbone with short acyl groups, *Brucella* spp. contain a modified lipid A layer that consists of a diaminoglucose backbone with long branching acyl groups [2]. This deviation from a consistent molecular structure has the potential to subvert immune recognition by the innate immune

system through complement interference and decreased cytokine production, leading to enhanced *Brucella* replication and pathogenesis. Despite its mechanisms of immune avoidance, there are some aspects of *Brucella* spp. that are recognized by the innate immune system, making the understanding of these mechanisms essential for targeting future treatments for brucellosis.

As hypothesized by Janeway (1989), the innate immune system has evolved over time to recognize consistent molecular structures in pathogens known as Pathogen or Damage-Associated Molecular Patterns (PAMPs or DAMPs). These PAMPs and DAMPs are recognized by protein structures known as pattern recognition receptors (PRRs) [4]. From previous studies, *Brucella* genomic DNA (gDNA) is known to be recognized by the PRR absent in melanoma 2 (AIM2) and subsequently promotes inflammation, making it an excellent PAMP for immune recognition [5–7]. PRRs include membrane-bound receptors, which consist of Toll-like receptors (TLRs) and C-type Lectin receptors (CLRs), as well as cytosolic receptors made up of a Nucleotide-Binding Domain and Leucine-Rich Repeat Containing receptors (NLRs), Aim-2-Like receptors (ALRs), Rig-I-Like Helicase receptors (RLRs), and the X-LR class of uncategorized receptors [8,9]. After the recognition of a PAMP or DAMP, PRRs generally serve as sca ffolding proteins to promote the initiation or inhibition of immune signaling pathways [9]. Of the PRRs that have been described in brucellosis, the best characterized have been the TLRs. From previous literature, many TLRs have been implicated with *Brucella* detection, which plays a role in bacterial signaling, host resistance, and dendritic cell activation [10–16]. TLRs also play important roles in the transcriptional generation of inactive inflammatory cytokines in response to *Brucella* infections that can be activated by NLR or ALR immune signaling complexes [17,18]. This indicates that multiple PRRs work in tandem to attenuate brucellosis pathogenesis.

Inflammasome-forming NLRs and AIM2 have also been reported to play a role in *Brucella* sensing [5–7]. After recognition, the NLR or ALR is able to bind the apoptosis-associated speck-like protein containing a caspase activation recruitment domain (CARD) (ASC) and procaspase-1 to form the canonical inflammasome [8]. The inflammasome then cleaves caspase-1, which subsequently cleaves the cytokines pro-IL-1β and pro-IL-18, produced through TLR signaling, to their active forms to promote inflammation [8,9,19–23]. Inflammasome signaling can also lead to a form of inflammatory cell death known as pyroptosis. Pyroptosis occurs when activated capase-1 cleaves the protein gasdermin D, releasing the gasdermin N subunit [24]. This subunit binds with phosphoinositides on the cell membrane and oligomerizes, creating membrane pores that lead to an osmotic imbalance in the cell that eventually leads to cell lysis [24]. Recently, the formation of a non-canonical inflammasome has been described that utilizes caspase-11 to mediate the cleavage of gasdermin D to initiate pyroptosis.

Previous studies evaluating inflammasome activation in response to *Brucella* have predominately focused on characterizing the activation of inflammatory cytokine signaling associated with canonical inflammasome activation. The best described inflammasomes involved in *Brucella* infections are NLR Family Pyrin Domain Containing 3 (NLRP3) and AIM2. In mouse models, the NLRP3 inflammasome promotes survival and decreased bacterial load through enhanced cytokine secretion, in addition to sensing mitochondrial reactive oxygen species (ROS) generated from *Brucella* [6,7]. Looking at the *Brucella* PAMPs, AIM2, as a known sensor of bacterial DNA, becomes activated from *Brucella* gDNA recognition and initiates inflammatory cytokine signaling and pyroptosis [5,6,25]. These inflammasomes are ASC-dependent, as shown in the formation of punctate ASC structures during infection [6], indicating that ASC-dependent inflammasomes are important in *Brucella* recognition and targeting through inflammatory cytokine signaling. Despite these advancements in studying inflammasome-mediated inflammatory cytokine signaling, pyroptosis and the role of gasdermin D have not been extensively evaluated in response to *Brucella.*

In this study, we used *Asc*−/− and *Caspase-11*−/− mice to further elucidate the role of the canonical and non-canonical inflammasomes following *B. abortus* infection. We sought to assess survival, histopathology, bacterial load, and cell death to provide a more holistic view of cytokine responses and pyroptosis, both in vivo and *in vitro*. Additionally, we reassessed *Brucella* PAMPs using *B. abortus* gDNA and LPS to better define the mechanisms associated with pathogen recognition. Ultimately, we

found that ASC functions to attenuate *B. abortus* pathogenesis through the modulation of inflammation and pyroptosis, requiring gasdermin D through a mechanism independent of caspase-11. Additionally, we determined that *Brucella* gDNA, rather than LPS, provoked an elevated inflammasome response that augmented pyroptosis. This report contributes to the current literature and provides some additional novel insights into potential mechanisms of inflammasome activation during brucellosis.
