Myeloid miR-155 plays a limited role in antibacterial defense during Klebsiella-derived pneumosepsis and is dispensable for lipopolysaccharide- or Klebsiella-induced inflammation in mice

Abstract MicroRNA-155 (miR-155) plays a crucial role in regulating host inflammatory responses during bacterial infection. Previous studies have shown that constitutive miR-155 deficiency alleviates inflammation while having varying effects in different bacterial infection models. However, whether miR-155 in myeloid cells is involved in the regulation of inflammatory and antibacterial responses is largely elusive. Mice with myeloid cell specific miR-155 deficiency were generated to study the in vitro response of bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and peritoneal macrophages (PMs) to lipopolysaccharide (LPS), and the in vivo response after intranasal or intraperitoneal challenge with LPS or infection with Klebsiella (K.) pneumoniae via the airways. MiR-155-deficient macrophages released less inflammatory cytokines than control macrophages upon stimulation with LPS in vitro. However, the in vivo inflammatory cytokine response to LPS or K. pneumoniae was not affected by myeloid miR-155 deficiency. Moreover, bacterial outgrowth in the lungs was not altered in myeloid miR-155-deficient mice, but Klebsiella loads in the liver of these mice were significantly higher than in control mice. These data argue against a major role for myeloid miR-155 in host inflammatory responses during LPS-induced inflammation and K. pneumoniae-induced pneumosepsis but suggest that myeloid miR-155 contributes to host defense against Klebsiella infection in the liver.


Introduction
Macr opha ge r esponses during bacterial infections and inflammatory conditions are regulated at multiple levels (Zhang andCao 2019 , 2021 ).MiR-155 is an important noncoding RNA that is induced during the macr opha ge inflammatory r esponse as a common target of a broad range of toll-like receptor (TLR) agonists and inflammatory mediators, such as tumor necrosis factor (TNF) (O'Connell et al. 2007, Chen et al. 2021 ).MiR-155 is induced in activ ated macr opha ges by dir ect binding of NF-κB to the miR-155 gene promoter following TLR activation.MiR-155 acts as a positive feedback signal to inflammatory responses by repressing the negative regulators SH-2 containing inositol 5' polyphosphatase 1 (SHIP1) and suppressor of cytokine signaling 1 (SOCS1), or suppressing interleukin (IL)-10 production (Billeter et al. 2014, Doxaki et al. 2015, Mann et al. 2017 ).In vivo data have indicated that miR-155 promotes lipopol ysacc haride (LPS)-induced acute lung injury in mice and rats (Wang et al. 2016 ) and that miR-155 overexpression in mice further enhances bacterial infection-induced inflammation (Yang et al. 2021 ).
Although the role of miR-155 in inflammatory responses caused by TLR agonists has been intensiv el y studied and welldocumented, its effect on host antibacterial defense is less explored.Both beneficial and detrimental effects of miR-155 on macr opha ge-mediated antibacterial r esponses hav e been r eported.MiR-155 contributes to enhanced phagocytosis of Streptococcus (S.) pneumoniae and Staphylococcus (S.) aureus by peritoneal macr opha ges (PMs; Yao et al. 2017 ), and miR-155 promotes the bactericidal capacity of the macr opha ge RAW264.7 cell line against S. aureus (Xu et al. 2013 ).Furthermore, miR-155 was r equir ed for clearance of S. pneumoniae colonization in a murine model (Verschoor et al. 2014 ).Conversely, miR-155 was documented to interfere with the bactericidal mechanisms in macr opha ges .For instance , miR-155 suppr essed macr opha gemediated bacterial phagocytosis and intracellular killing of Pseu-domonas aeruginosa (Yang et al. 2014 ).Mor eov er, miR-155 inhibited apoptosis by destabilizing Casp-3 mRNA in RAW 264.7 macr opha ges (De Santis et al. 2016 ), thereby providing a niche that favors replication of intracellular bacteria, such as Mycobacterium tuberculosis , within infected macr opha ges (Rothc hild et al. 2016 ).Furthermor e, miR-155 incr eased mortality and impaired bacterial clearance of S. aureus in a postviral bacterial pneumonia model (Podsiad et al. 2016 ).
Klebsiella (K.) pneumoniae is a common causative pathogen in hospital-acquired pneumonia and sepsis (Mayr et al. 2014, Rudd et al. 2020 ).Our pr e vious studies r e v ealed that host defense a gainst K. pneumoniae infection is dependent on TLR2 and TLR4 and expression of the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) in myeloid cells (Branger et al. 2004, Wieland et al. 2011, van Lieshout et al. 2014 ).In view of the importance of miR-155 in TLR ligand-induced macr opha ge inflammatory r esponses, we sought to determine the contribution of myeloid cell specific miR-155 to the host response during LPS-induced lung inflammation and during pneumosepsis caused by K. pneumoniae using mice with a myeloid-specific miR-155 deficiency and well-established models (de Stoppelaar et al. 2014 ).

Animals
Homozygous Mir155 fl/fl mice (stocknumber 026700, The Jackson Laboratory) (Hu et al. 2014 ) were crossed with LysM Cre mice (Clausen et al. 1999, van Lieshout et al. 2014 ) to generate myeloid cell specific miR-155-deficient ( Mir155 fl/fl LysM Cre ) mice.Mir155 fl/fl Cr e-negativ e littermates ( Mir155 fl/fl mice) were used as controls in all experiments.All genetically modified mice were backcrossed at least eight times to a C57Bl/6 bac kgr ound and age and sex matched when used in experiments.Mice were used at 8-12 weeks of age.All mouse experiments were approved by the Institutional Animal Care and Use Committee of the University of Amsterdam.
PM isolation and stimulation.PMs were harvested by peritoneal lav a ge and seeded in 48-well flat bottom culture plates (Greiner bio-one) at a density of ∼0.5 × 10 6 cells per well in CM and left to adhere for 3 hours (Qin et al. 2021 ).PMs were then washed intensiv el y and stim ulated with 100 ng/ml ultr a pur e LPS for 6 or 24 hours.Cell supernatant was collected and stored at −20 • C until further anal ysis, cells wer e harv ested in TRizol (Invitrogen) and stored in −80 • C for RNA isolation.
Alveolar macrophage (AM) isolation and stimulation.AMs were harvested by broncho-alveolar lavage (BAL) using a well-established method (Busch et al. 2019 ) providing pure and functional cells (Otto et al. 2022 ), and seeded in 96-wells flat bottom culture plates (Greiner bio-one) at a density of ∼5 × 10 4 cells per well in CM and left to adhere for 3 hours (Qin et al. 2021 ).AMs wer e stim ulated with 100 ng/ml of ultr a pur e LPS for 6 hours.Cell supernatant was collected and stored at −20 • C until further analysis and cells were harvested in TRizol (Invitrogen) and stored in −80 • C for RNA isolation.

Quantitati v e rev erse transcription PCR (qRT-PCR)
Total RN A w as extracted b y using TRizol (Invitrogen) accor ding to the manufacturer's instruction.To measure miRNA expression, cDN A w as synthesized with TaqMan Micr oRNA Re v erse Tr anscription Kit (Thermo Fisher).TaqMan miRNA Assays (Applied Biosystems) for matur e mm u-mir-155 (Assay ID; 0002571) were performed using a Roche LightCycler 480 thermocycler.Data were analyzed with LinRegPCR based on PCR efficiency values derived from amplification curves (Ramakers et al. 2003 ).Relativ e expr ession of Mir155 was normalized to U6 snNA (Assay ID; 001973).

Mouse models
Lung inflammation and pneumonia were induced as previously described (Qin et al. 2022 ).Briefly, mice wer e administr ated intr anasall y with 1 μg of ultr a pur e LPS in 50 μl saline (Otto et al. 2020 ) and euthanized 6 hours postinoculation.The right lung was used for BAL and the left lung was pr eserv ed for histopathology after fixation in 10% formalin.Cell counts in BAL fluid (BALF) were determined using a hemocytometer (Coulter) and different cell populations in BALF samples were determined by flow cytometry (details see below) (de Porto et al. 2019 ).BALF supernatants were stored at −20 • C until further analysis.
Pneumonia was induced by intranasal inoculation of ∼10 4 CFU K. pneumoniae serotype 2 (American Type Culture Collection number 43816).At various time points, BALF and lung tissues were collected in the same way as described for LPS-induced lung inflammation.Blood was collected in heparin minicollect tube (Greiner Bio-One) for direct analysis of bacterial loads and leukocytes or for isolation of plasma and stored at −20 • C until further analysis.Spleens and livers were collected and homogenized for bacterial loads or fixed for histopathology.Bacterial loads in blood and tissues homogenates were determined by counting CFU from serial dilutions plated on blood agar plates, incubated at 37 • C for 16 hours.Cells in B ALF w er e anal yzed as described belo w.B ALF supernatant and plasma were subjected to ELISA or CBA.BALF samples and blood were also collected from naïve mice.
P eritonitis w as induced b y intr a peritoneal injection of 2 mg/kg ultr a pur e LPS as pr e viousl y described (Qin et al. 2021 ) and mice were sacrificed 3 hours later.PLF was isolated for analysis of cells (as described above) or stored at −20 • C until further analysis.

Histology
P ar affin embedded lungs and liv ers wer e cut into 4μm sections and stained with hematoxylin and eosin (H&E).Lung inflammation was scored by a pathologist blinded for treatment and genotype of the mice as pr e viousl y described (Meijer et al. 2021 ) for the following par ameters: br onc hitis, edema, interstitial inflammation, intr a-alv eolar inflammation, pleuritis , endothelialitis , and percentage of the lung surface demonstrating confluent inflammatory infiltr ate.Eac h par ameter was gr aded 0-4, with 0 being "absent" and 4 being "se v er e;" the total pathology score was expressed as the sum of the score for all parameters with a maxim um scor e of 32.Liv er pathology was scor ed as pr e viousl y described (Claushuis et al. 2018 ) for inflammation, necrosis, and presence of thrombi, with a maximum score of 12.

Sta tistical anal ysis
Nonpar ametric v ariables wer e anal yzed using the Mann-Whitney U-test.Analysis was done using Gr a phP ad Prism version 8 (Gr a phpad Software, San Diego, CA).Statistical significance is shown as * P < .05;* * P < .01.

Myeloid miR-155 deficiency does not affect immune cell development in mice
While miR-155 is known to be involved in the regulation of inflammation during infection and sepsis (Chen et al. 2021 ), the role of miR-155 in myeloid cells, the major class of immune effector cells during infection, is less well-explored.T hus , we here sought to determine the role of myeloid miR-155 in vitro and in vivo during inflammation induced by the Gr am-negativ e bacterial cell wall component LPS or by K. pneumoniae infection.To this end, we generated myeloid cell specific miR-155-deficient mice ( Mir155 fl/fl LysM Cre ) and littermate controls ( Mir155 fl/fl ).In AMs, PMs and BMDMs of Mir155 fl/fl LysM Cre mice, miR-155 expression was decreased by 72%, 80%, and 65%, r espectiv el y, compar ed to cells of Mir155 fl/fl littermates ( Figure S1A, Supporting Information ).As previous studies r e v ealed that miR-155 deficiency affects the de v elopment of immune cells (Kohlhaas et al. 2009, Mann et al. 2017, Pashangzadeh et al. 2021 ), we assessed whether deficiency of miR-155 in myeloid cells impacted on myeloid populations in the alveolar lumen of the lung and in blood.AMs are the dominant cell population in the lung lumen, while neutrophils and monocytes numbers ar e v ery low.No differ ences wer e found in these cell populations between naïve Mir155 fl/fl LysM Cre mice and littermate contr ols ( Figur e S1B, Supporting Information ).Like wise, the myeloid cell populations in blood were also not affected by miR-155 deficienc y in my eloid cells ( Figure S1C, Supporting Information ).Taken together, these data indicate that miR-155 deficiency in the myeloid lineage does not affect myeloid cell numbers in the lung and circulation.

Myeloid miR-155 deficiency represses TLR ligands-induced macr opha ges activ a tion in vitro
Since miR-155 is known to regulate inflammatory responses during infections (Chen et al. 2021 ), we investigated the role of miR-155 in cytokine secretion by macr opha ges in vitro in response to various TLR ligands.To this end, we generated BMDMs and isolated primary AMs and PMs of Mir155 fl/fl LysM Cre and Mir155 fl/fl control mice and stimulated these with LPS (TLR4 ligand), PAM3CSK4 (TLR1/2 ligand), LTA (TLR2 ligand), or poly(I:C) (TLR3 ligand).In miR-155-deficient BMDMs, secretion of both IL-6 and TNF was decreased at either 6 or 24 hours after stimulation with all these TLR ligands, as compared to wild type BMDMs (Fig. 1 A-D).Compared to wild type AMs, miR-155-deficient AMs released significantly less proinflammatory cytokines IL-6 and TNF, and chemokines CXCL1 and CXCL2 in response to LPS (Fig. 1 E).
Likewise, PMs of Mir155 fl/fl LysM Cre mice secreted less IL-6 and TNF after stimulation with LPS when compared to PMs from the Mir155 fl/fl control mice (Fig. 1 F).These results indicate that myeloid specific deficiency of miR-155 attenuates inflammatory responses in macr opha ges upon activ ation by TLR ligands in vitro , corr obor ating pr e vious findings fr om in vitro experiments with cells fr om constitutive miR-155-deficient mice (Taganov et al. 2006, Mann et al. 2017 ).

Myeloid miR-155 deficiency does not affect LPS-induced inflammation in vivo
To determine the role of myeloid miR-155 in lung inflammation in vivo , we first investigated the inflammatory response in the lung of mice challenged with LPS via the airwa ys .Analysis of BALF for hallmark inflammatory reactions revealed that, opposite to our in vitro results, myeloid specific miR-155 -deficient mice displayed comparable levels of IL-6, TNF, CXCL1, and CXCL2 during LPS-induced lung inflammation (Fig. 2 A).Furthermor e, leuk ocyte numbers and neutrophil influx (Fig. 2

Myeloid miR-155 deficiency impacts host defense in the li v er but does not affect systemic inflammatory responses during Klebsiella pneumosepsis
Since the K. pneumoniae strain used in our study can disseminate and cause systemic inflammation and organ damage (Claushuis et al. 2016 ), we next analyzed the bacterial burden in blood and distant organs (spleen and liver) at 16 or 44 hours postinfection.Bacterial loads in the liver of Mir155 fl/fl LysM Cre mice were significantly higher than in littermate controls at 44 hours after infection, while in blood and spleen a similar trend was observed (Fig. 4 A).To gain insight into the effect of myeloid miR-155 on systemic inflammatory r esponses, we measur ed le v els of IL-6, TNF, and CCL2 le v els in plasma from Mir155 fl/fl LysM Cre and control mice.None of the mediators were affected by myeloid miR-155 deficiency (Fig. 4 B).Likewise, neither neutrophil numbers nor neutrophil activation (CD11b expression) in blood were altered by miR-155 deficiency in myeloid cells ( Figure S2, Supporting Information ).In view of the higher bacterial loads in the liver of Mir155 fl/fl LysM Cre mice, we examined liver pathology and plasma le v els of AST, ALT (reflecting hepatocellular injury).Histopathological changes in liver were equal between Mir155 fl/fl LysM Cre and Mir155 fl/fl mice (Fig. 4 C).Measurement of plasma AST and ALT, as well as LDH (indicative of cellular injury in general) sho w ed that these parameters were increased at 44 hours postinfection and were slightly but not significantly lo w er in myeloid miR-155deficient mice at this time point (Fig. 4 D).Together these data suggest that myeloid miR-155 contributes to host defense in the liver but may promote liver damage while being dispensable for regulation of systemic inflammatory responses during K. pneumoniaederi ved pneumose psis .

Discussion
MiR-155 is an important regulatory molecule for inflammatory responses in multiple cell types.We here investigated its function in myeloid cells, crucial components of the imm une r esponse during bacterial infection, both in vitro and in vivo .Our data indicates that miR-155 potentiates inflammatory responses of macrophages in an in vitro setting, with reduced cytokine secretion by miR-155deficient macr opha ges after stim ulation with m ultiple TLR ligands, while myeloid miR-155 deficiency had no effect on inflammatory responses in vivo during LPS-induced inflammation or intact K. pneumoniae infection.Inter estingl y, we found that miR-155 might play a role in host defense in the liver during Klebsiella pneumosepsis.
Ov er all genetic depletion of miR-155 leads to abnormalities in the de v elopment of the imm une system (Kohlhaas et al. 2009, Mann et al. 2017, Pashangzadeh et al. 2021 ).Unlike most pr e vious studies with constitutive miR-155 -deficient mice, we here used myeloid cell specific miR-155-deficient mice that are phenotypically normal and sho w ed an unaltered immune cell de v elopment.Our in vitro data sho w ed that miR-155 potentiates TLR ligand-induced inflammatory mediator production in macr opha ges, whic h is in a gr eement with results from previous studies using mice with constitutive deficiency of miR-155 (Mann  et al. 2017 ).Strikingly, we found that, opposite to our findings with AMs and PMs in vitro, wherein miR-155 deficiency was associated with reduced IL-6, TNF, CXCL-1, and CXCL-2 production, upon in vivo targeting of the sites (lung or peritoneal cavity) where these tissue macr opha ges r eside miR-155 deficienc y w as not associated with lo w ered c ytokine responses .T he most likely explanation for the discrepanc y betw een these in vivo and in vitro data is the contribution of different cell types besides macrophages to the production of these mediators in animals.Additionally, miR-155 can be produced and secreted by other nonimmune cells (Zheng et al. 2017, Jiang et al. 2019 ), thus miR155 signalling is likely to be functionally maintained in macrophages of Mir155 fl/fl LysM Cre mice in vivo .Hence, although miR-155 potentiates the production of various cytokines in tissue macr opha ges fr om differ ent or gans, its deficiency in macr opha ges in mice in vivo seems to have a lesser effect on host inflammatory responses.
Myeloid cells, via TLR-dependent signaling, play an important role in antibacterial defense during K. pneumoniae infection (Branger et al. 2004, Wieland et al. 2011, van Lieshout et al. 2014 ).Pr e vious studies hav e indicated that TLR activ ation induces the expression of miR-155 expression in macrophages (O'Connell et al. 2007 ).In a gr eement with this, miR-155 expression was increased in the murine AM cell line RAW264.7 after infected with K. pneumoniae (Teng et al. 2016 ).Furthermore, miR-155 was suggested to augment phagocytosis of S. pneumoniae and S. aureuvs by macr opha ges in a TLR2-dependent manner (Yao et al. 2017 ).Additionall y, miR-155 pr omotes neutr ophil extr acellular tr a p formation, which is known as a crucial mechanism for controlling K. pneumoniae infection in mice (Claushuis et al. 2018, Hawez et al. 2022 ).All these mechanisms may explain our finding that the bacterial burden in myeloid miR-155 -deficient mice was higher in liver than in littermate control mice.Recently, it was reported that Kupffer cells in the liv er, the lar gest population of tissue r esident macr opha ges, can sequester hypervirulent Klebsiella intr acellularl y and provide a niche for bacterial persistence (Wanford et al. 2021 ).Further investigation, howe v er, is r equir ed to determine the mechanism by which miR-155 mediates antibacterial defense against Klebsiella in the liver and whether miR-155 in Kupffer cells is involved in phagocytosis, killing or sheltering of Klebsiella .
Pneumonia is the leading cause of sepsis (Torres et al. 2021 ), which is defined as a life-threatening organ dysfunction caused by a dysregulated host response to an infection (Singer et al. 2016 ).
Emer ging e vidence fr om both clinical inv estigations and m urine experiments has r e v ealed that miR-155 is involved in organ damage during sepsis (Liu et al. 2015, Hawez et al. 2022 ).MiR-155 was significantl y upr egulated in blood leuk ocytes of sepsis patients (Liu et al. 2015 ).Plasma miR-155 le v els wer e ele v ated in patients with septic cardiac dysfunction (Wang et al. 2016 ) and indicative of a more severe condition and poorer prognosis (Liu et al. 2015 ).
Ther efor e, a high le v el of miR-155 was suggested as a potential biomarker for predicting mortality and treatment outcome of sev er e sepsis in patients (Liu et al. 2015, Han et al. 2016 ).The same phenotype was ca ptur ed in mouse sepsis models.MiR-155 expression was significantly higher in septic mice and associated with accelerated lung and liver injury (Tuerdi et al. 2018, Yang et al. 2018 ), while the inhibition of miR-155 attenuated sepsis-induced liver and lung damage and increased the survival rate of septic F igure 4. My eloid miR-155 deficiency impacts host defense in the liver and bacterial dissemination but does not affect systemic inflammatory responses during Klebsiella pneumosepsis.Mir155 fl/fl LysM Cre and control Mir155 fl/fl mice were infected with 10 4 K. pneumoniae via the airways for 16 or 44 hours.(A) Bacterial burden (CFU) in liver, spleen, and blood.(B) IL-6, TNF, and CCL-2 protein levels in plasma.(C) Liver pathology scored according to the semiquantitative scoring system described in the section "Methods."(D) Organ damage parameters AST , ALT , and LDH le v els in plasma.N = 8.Data are shown as bar graphs with mean ± SD with individual values.P-values were calculated using Mann-Whitney test.* P < .05. mice (Lv et al. 2015, Tuerdi et al. 2018, Yang et al. 2018 ).In the present study, we found that organ damage parameters (LDH, ALT, and AST) sho w ed a tendenc y for lo w er le v els in myeloid miR-155deficient mice after infection with K. pneumoniae, suggesting that miR-155 in myeloid cells is sufficient to augment sepsis-induced or gan dama ge .T hese findings , ho w e v er, wer e not corr obor ated by significant histopathological changes in the liver (or lung).A possible reason for this discrepancy might be that the K. pneumoniae infection causes mild histopathological changes in the lung and liver, as sho wn b y lo w pathological scores in both groups even at 44 hours after infection, that are not sufficient to distinguish a role of miR-155 by this method.
As a limitation of our study, it is worth noting that the bacterial strain used in this study is a mouse-adapted strain, and the host responses may vary significantly with different bacterial str ains (Bengoec hea and Sa Pessoa 2019 ).Mor eov er, the outcome of the infection can be notably influenced by specific strains of Klebsiella (Wanford et al. 2021 ).Ther efor e, we cannot rule out that miR-155 deficiency in myeloid cells might have a different impact on the host's immune response when encountered by another Klebsiella str ain.Further inv estigation is warr anted to understand the impact of miR-155 deficiency on the host immune responses a gainst v arious str ains of Klebsiella .Further studies with purified macr opha ges fr om affected or gans ar e also r equir ed to elucidate the a ppar ent discr epanc y betw een in vitro and in vivo cytokine r esults, since infiltr ating cells and par enc hymal cells may contribute to cytokine le v els in vivo .Another limitation of our study is that the le v els of miR-155 in macr opha ges of Mir155 fl/fl LysM Cre mice did not result in complete reduction, which could explain the limited effect of the miR-155 deficiency.Pr e viousl y, we hav e shown that LysM Cre lar gel y, but not completel y r educed pr otein le v els of the targeted genes in AMs , PMs , and BMDMs (Anas et al. 2016, de Porto et al. 2021, Otto et al. 2022 ).In the current study, we used a TaqMan miRNA assay to analyze the expression of the target gene, which may be more sensitive for residual expression in LysM Cre targeted cells.Another explanation for miR-155 in these cells may be uptake of miR-155 carrying micr ov esicles deriv ed fr om other cells (Gomez et al. 2020 ).
In conclusion, we here used Mir155 fl/fl LysM Cre mice to assess the role of myeloid cell-specific miR-155 and show that miR-155 acts B), as well as neutrophil activation (as indicated by CD11b expression and MPO levels in BALF) (Fig.2C and D) were also not affected by miR-155 deficiency in myeloid cells.In view of the robust expression of miR-155 in PMs ( FigureS1A, Supporting Information ), we also investigated the role of myeloid miR-155 in the inflammatory response in the peritoneal cavity after intr a peritoneal injection of LPS.Similar to LPSinduced lung inflammation, LPS-induced peritonitis in myeloid miR-155-deficient mice was not associated with an altered cytokine and chemokine response (Fig.2 E); Similarly, the neutrophil influx (Fig.2 F), as well as neutrophil activation (as indicated by CD11b expr ession, Fig.2 G) wer e also compar able betw een my eloid miR-155-deficient mice and littermate controls.Taken together these results indicate that miR-155 in myeloid cells does not impact on innate immune responses during acute LPS-induced inflammation in vivo .

F igure 3 .
My eloid miR-155 deficiency does not impact on host defense and inflammatory responses in the lung during K. pneumonia.Myeloid miR-155-deficient mice ( Mir155 fl/fl LysM Cre ) and control mice ( Mir155 fl/fl ) were infected with 10 4 K. pneumoniae via the airways for 16 or 44 hours.(A) Bacterial burden (CFU) in lung, (B) IL-6 and TNF le v els, (C) neutr ophil influx (PMNs), and (D) neutr ophil CD11b expr ession and MPO le v els in BALF.(E) Lung pathology scored according to the semiquantitative scoring system described in the Methods.N = 8.Data are shown as bar graphs with mean ± SD and individual values.Data between Mir155 fl/fl LysM Cre and control Mir155 fl/fl mice were not significantly different.