INTRODUCTION NR4A1 in K. pneumoniae Lung Infection 4 rapidly induced during infection, and that Nr4a1 -/mice had decreased bacterial burden in vivo, 70 though the mechanisms underlying these observations remain unclear (21) .
The current study examined the role of NR4A1 in K. pneumoniae pneumonia. Here, we found 73 that Nr4a1 deficiency led to an increase of K. pneumoniae bacterial lung burden and 74 dissemination. Mechanistically, phagocyte recruitment was unaffected, though inflammatory 75 cytokine/chemokine production was decreased in Nr4a1 -/mice, identifying that Nr4a1 -/-76 neutrophils had defective bactericidal function and attenuated inflammatory gene expression, 77 suggesting that Nr4a1 is a novel, critical mediator of neutrophil function. hours after infection in the lung, as a measure of local control, and in the spleen as a measure of 84 bacterial dissemination. Compared to WT mice, Nr4a1 -/mice had significantly increased K. 85 pneumoniae lung burden and spleen burden 48 hours post-infection ( Fig. 1c-d) . These 86 differences were not present at 24 hours post-infection ( Fig. 1a-b) . These data identify that 87 Nr4a1 -/mice controlled K. pneumoniae acute lung infection poorly, suggesting that Nr4a1 may 88 play an important role in the immune response to acute K. pneumoniae lung infection. Cxcl2, were significantly decreased in the BALF of Nr4a1 -/mice ( Fig. 2a, b, e) . However, Tnfa 97 and the chemokines Cxcl1 and Cxcl5 were not different (Fig. 2c , d, f), suggesting the NR4A1-98 dependent gene regulation was reflective of a specific cell population or pathway mediating the 99 phenotype and this was observable within 5 hours post-infection. whether the Nr4a1-dependent phenotype was associated with reduced production of the effector 106 molecule myeloperoxidase (MPO), an enzyme critical for phagocyte-mediated bacterial killing 107 of K. pneumoniae (22). In the Nr4a1 -/strain, MPO production trended down within 5 hours 108 post-infection (P = 0.0689) (Fig. 3b ). By the 24 hours post-infection timepoint, Nr4a1 -/mice 109 had significantly decreased MPO protein in the lungs compared to WT mice (Fig. 3d) . These 110 data suggest that early immune gene transcription reflects protein production during K. 111 pneumoniae lung infection, and that Nr4a1-deficient susceptibility to infection is associated with 112 reduced production of phagocyte markers. To examine a potential mechanism by which Nr4a1 protects against lung infection, we 117 performed flow cytometry on the lungs of WT and Nr4a1 -/mice at 24 hours post K. pneumoniae 118 infection, prior to differences in bacterial burden, to determine if Nr4a1 deletion resulted in a 119 change in immune cell recruitment. No significant differences were observed in inflammatory 120 macrophages (CD45 + /CD11b + /CD11c + ), alveolar macrophages (CD45 + /CD11c + /SiglecF + ), or 121 neutrophils (CD45 + /CD11b + /Ly6G + ) ( Fig. S1b-d) . Interestingly, no differences in transcript 122 were found between pro-inflammatory cytokines Il1b, Tnfa, or Il6, or the neutrophil-specific 123 gene Lcn2 (Fig. S2a-d) . These data suggest the role Nr4a1 plays in the immune response to K. Neutrophil bacteria killing assays were similarly analyzed to determine bacterial growth as a 137 measure of in vitro bactericidal function from WT and Nr4a1 -/mice. Notably, in WT 138 neutrophils co-cultured with K. pneumoniae, Nr4a1 gene transcription is induced compared to 139 naïve WT neutrophils (Fig. 4a) . Supernatants from Nr4a1 -/neutrophils had significantly higher 140 K. pneumoniae growth compared to WT neutrophils within 60 minutes, though at 30 minutes no 141 differences were observed between genotypes (Fig. 4b-c) . These data suggest that Nr4a1 -/-142 neutrophils K. pneumoniae killing is time dependent. Taken together, these data suggest Nr4a1 To assess Nr4a1-dependent transcriptional changes in neutrophils, Nr4a1 -/neutrophil RNA was 148 isolated 30 minutes after K. pneumoniae co-culture (when the bacterial burdens are equivalent).
Here, Nr4a1 -/neutrophils had decreased Il1b transcript compared to WT neutrophils, as was The current study aimed to determine the role of NR4A1 in K. pneumoniae pulmonary infection.
Prior studies have described a limited or detrimental role for NR4A1 in bacterial infections (19, relevance is limited, as E. coli is not a cause of community or hospital acquired pneumonia, and 183 little mechanistic information was ascertained from this work. Conversely, our study determined 184 that Nr4a1 loss was detrimental in the K. pneumoniae pneumonia model. Mechanistically, the 185 observation that NR4A1 deficiency did not impair macrophage or neutrophil cell recruitment to 186 the lung suggested that NR4A1 may be important for innate antibacterial cell-intrinsic function.
While some genes were unchanged, reduced lung expression of the innate pro-inflammatory Further study into the use of NR4A1 as a potential therapeutic target for host-immunity 227 responses is also warranted. While the present study shows the deletion of Nr4a1 to be 228 NR4A1 in K. pneumoniae Lung Infection 11 detrimental to murine clearance of K. pneumoniae pneumonia through loss of neutrophil 229 function, further study of the ability to rescue or enhance NR4A1 function is arranged in order to 230 determine this potential, including the use of the NR4A1 agonist, cytosporone B, or the NR4A1 231 ligand-binding chemical, PDNPA, in the context of K. pneumoniae (11, 17, 40) .
The current study also only looked at the role of NR4A1 in K. pneumoniae pneumonia. While K. 234 pneumoniae is a prominent cause of pneumonia, it is not the only bacteria to do so, nor does K. The current study illustrates that Nr4a1 is an important component in host immune response to 242 K. pneumoniae pulmonary infection, specifically in neutrophils, marking NR4A1 as a potential 243 target for future immunotherapies. Finding potential immunotherapy targets against bacterial 244 infections is an increasing concern for the public health community as antibiotic-resistant strains 245 continue to increase. Demonstrating the role NR4A1 has in host immune response to K. 246 pneumoniae provides a rational biologic target around which to develop immunotherapies.
Additionally, the novel finding that Nr4a1 is an essential factor in neutrophil-specific response to For BALF, 9-week-old male mice were inoculated with 5x10 3 Klebsiella pneumoniae CFUs.
After 5 hours, the mice were euthanized under isoflurane and a secondary terminal bleed and 288 BALF was acquired by flushing the lungs with 1mL sterile 1XPBS. The 5-hour timepoint was 289 selected based on prior LPS-induced acute lung injury (ALI) data suggesting neutrophil influx to 290 the lungs starts as early as 2 hours after insult and as early as 3 hours after a gram-negative 291 bacterial insult (41) (42) (43) . Once counted, cells were overlaid on a 40%/70% Percoll (Fisher Scientific, Waltham, MA) 345 gradient and centrifuged for 30 minutes at 427xg and 28˚C. After 30 minutes, neutrophils were 346 collected from the interface of the 40% and 70% Percoll layers, washed twice with 347 unsupplemented RPMI 1640 and spun at 427xg and 4˚C for 7 minutes, and resuspended in 1mL 348 PBS. Neutrophils were counted and then plated in a 96-well plate at 2.5x10 5 neutrophils/well.
Neutrophils were incubated at 37˚C and 5.0% CO2 for a 1-hour recovery period before being 350 used in a bacterial killing assay. 
The authors declare no conflict of interest. 