SKAP2 is required for defense against K. pneumoniae infection and neutrophil respiratory burst
Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that can cause a wide range of life-threatening infections , including pneumonia , sepsis and urinary tract infections ( Bengoechea and Sa Pessoa , 2019; Paczosa and Mecsas , 2016; European Centre for Disease Prevention and Control , 2018; Weiner et al . , 2016 ) . K . pneumoniae is a leading cause of hospital-associated infections with one recent study reporting that K . pneumoniae contributes to 7 . 7% of cases from over 4500 hospitals ( Weiner et al . , 2016 ) . The increasing number of severe , and sometimes systemic , K . pneumoniae infections are largely attributed to a rise in antibiotic-resistant strains ( Falagas et al . , 2014; European Centre for Disease Prevention and Control , 2018; Kobayashi et al . , 2016 ) and hypervirulent strains that generate thicker capsules ( Zhang et al . , 2016; Harada et al . , 2019; Lam et al . , 2018 ) . Highlighting the importance of the innate immune response , neutropenic patients are highly susceptible to life-threatening respiratory and bloodstream infections , including those caused by K . pneumoniae ( Zhu et al . , 2018; Micozzi et al . , 2017 ) . K . pneumoniae lung pathogenesis has been extensively investigated in a mouse model using the rodent-adapted ATCC 43816 ( Bengoechea and Sa Pessoa , 2019; Lawlor et al . , 2005; Xiong et al . , 2015; Bachman et al . , 2015; Vornhagen et al . , 2019; Batra et al . , 2012 ) . In murine models of infection , the K . pneumoniae 43816 strain robustly infects lungs eliciting a strong innate immune response through rapid and concurrent recruitment of neutrophils and iMOs to the infected lungs ( Lawlor et al . , 2005; Xiong et al . , 2015; Cai et al . , 2010; Batra et al . , 2012; Sharma et al . , 2014; Ye et al . , 2001 ) . Work using this strain has revealed several genes that protect K . pneumoniae against the host neutrophil response ( Paczosa et al . , 2020; Silver et al . , 2019 ) . Neutrophils are the first responder cell type for fighting against invading pathogens , however , their activation is tightly regulated to prevent severe tissue damage that have been correlated with several autoimmune and inflammatory diseases ( Mócsai , 2013 ) . At the site of infection , neutrophils can bind to pathogens leading to the activation of various killing mechanisms , including phagocytosis , generation of reactive oxygen species ( ROS ) , degranulation , and release of neutrophil extracellular traps ( NETs ) ( Futosi et al . , 2013; Nguyen et al . , 2017; Mócsai , 2013 ) . Although the K . pneumoniae capsule decreases bacterial binding and internalization ( Regueiro et al . , 2006; March et al . , 2013 ) , ROS , degranulation , NETs , and cytokine production have all been implicated in host defense against K . pneumoniae ( Bengoechea and Sa Pessoa , 2019; Chen et al . , 2016; Hirche et al . , 2005; Ivin et al . , 2017; Paczosa et al . , 2020; Papayannopoulos et al . , 2010; Zhao et al . , 2015 ) . The importance of ROS in host protection is highlighted in the setting of Chronic Granulomatous Disease ( CGD ) , a disease characterized by genetic mutations in the NADPH oxidase , where patients are unable to make ROS and are susceptible to Klebsiella infections ( Wolach et al . , 2017; Bortoletto et al . , 2015 ) . Furthermore , mice with defective ROS production ( Cybb-/- ) have a higher bacterial burden than wild-type mice during K . pneumoniae lung infection ( Paczosa et al . , 2020 ) . Receptor-mediated ROS production in neutrophils is driven by signal-transduction pathways that activate components of the NADPH oxidase complex ( Nguyen et al . , 2017; Futosi et al . , 2013 ) . These signaling pathways are tightly regulated to prevent inappropriate activation and subsequent tissue damage ( Futosi et al . , 2013 ) . Work in mice suggests that signaling through integrin , mincle receptors , G-protein couple receptors ( GPCRs ) and toll-like receptors ( TLRs ) are critical for protection against K . pneumoniae infection ( Teng et al . , 2016; Sharma et al . , 2014; Sharma et al . , 2017; Regueiro et al . , 2006; Galvão et al . , 2018; Bengoechea and Sa Pessoa , 2019 ) . In fact , multiple receptors may sense and/or bind to K . pneumoniae triggering ROS production . Prior studies have indicated a critical role for Src Kinase Associated Phosphoprotein-2 , SKAP2 ( SKAP-Hom and SKAP-55R ) downstream of integrin , and GPCRs in neutrophils and macrophages ( Boras et al . , 2017; Alenghat et al . , 2012; Swanson et al . , 2008; Tanaka et al . , 2016; Mócsai et al . , 2002; Togni et al . , 2005 ) . SKAP2 , a cytosolic adaptor protein , is required for integrin-mediated ROS production , adhesion , and migration to sterile inflammatory sites through its interaction with mediators of actin rearrangement ( Alenghat et al . , 2012; Boras et al . , 2017; Shimamura et al . , 2013; Tanaka et al . , 2016 ) . SKAP2 is also a target for inactivation by bacterial virulence factors , suggesting that it plays a key role in host defenses against infections ( Black et al . , 2000; Rolán et al . , 2013 ) . In humans , the Skap2 gene has been identified as a risk locus for Type 1 Diabetes , and Crohn’s disease ( Jostins et al . , 2012; Barrett et al . , 2009 ) . In resting cells , SKAP2 is thought to exist in an autoinhibitory conformation as a homodimer that is constitutively bound to PRAM-1 in neutrophils ( Moog-Lutz et al . , 2001; Rolán et al . , 2013 ) , or its homolog , ADAP , in other immune cells ( Liu et al . , 1998; Marie-Cardine et al . , 1998; Black et al . , 2000; Ophir et al . , 2013 ) . Upon receptor activation , SKAP2 changes to an active conformation that is localized to the plasma membrane , which allows it to interact other effector proteins including Src Family Kinases ( SFKs ) ( Bureau et al . , 2018; Marie-Cardine et al . , 1998; Liu et al . , 1998 ) . In addition , activated SKAP proteins can relay information from receptors to focal adhesion kinases ( FAK1/Pyk2 ) to other cytoskeletal mediators ( Ménasché et al . , 2007; Boras et al . , 2017; Tanaka et al . , 2016; Raab et al . , 2017; Ophir et al . , 2013 ) . Using a murine model of K . pneumoniae 43816 infection , we characterized the role of SKAP2 in pulmonary host defense against K . pneumoniae in this study . We demonstrate that SKAP2-expressing neutrophils contributed to the pulmonary containment of K . pneumoniae in lungs independent of inflammatory monocytes . Using murine neutrophils , we show that K . pneumoniae-induced ROS production requires SKAP2 , SFKs , Syk , PLCγ , Btk , and PKC , and that SKAP2 is required for maximal K . pneumoniae-activated phosphorylation of the tyrosine kinases SFKs , Syk , and Pyk2 .
To examine the role of SKAP2 in the pathogenesis of pneumonic K . pneumoniae infection , we intranasally infected wild-type ( WT ) BALB/c or BALB/c Skap2-/- mice with a streptomycin-resistant derivative of K . pneumoniae 43816 , a hyper-capsule producer that belongs to one of the most common hypervirulent serotype , K2 ( Paczosa and Mecsas , 2016; Paczosa et al . , 2020; Silver et al . , 2019 ) . Previous studies using K . pneumoniae 43816 or a derivative of this strain have reported a 50% lethal dose at 48 hr after infection with 3−5 × 103 colony forming units ( cfu ) in BALB/c mice , and a robust infection with high bacterial burden and cellular infiltration in the lungs by 24 hr with mice developing disseminated infection by 48 hr in C57BL/6J ( Lawlor et al . , 2005; Xiong et al . , 2015; Ye et al . , 2001; Feldman et al . , 2019 ) . WT and Skap2-/- mice were infected with 5 × 103 cfu and were monitored for weight loss , bacterial colonization , and innate cell recruitment at 8 , 16 , and 24 hr post infection ( hpi ) ( Figure 1A–C; Figure 1—figure supplement 1A , E–J ) . Consistent with prior studies , by 24 hr , infected WT mice lost 2% of their body weight , and K . pneumoniae reached on average 106 cfu with a 5-fold increase in neutrophil presence in the infected lungs ( Figure 1A–C; Figure 1—figure supplement 1A; Xiong et al . , 2015 ) . However , Skap2-/- mice lost significantly more body weight than the WT cohort at 24 hpi ( Figure 1A ) and had significantly higher bacterial loads in their lungs at 16 and 24 hpi ( Figure 1B ) , indicating that Skap2-/- mice are less competent at controlling K . pneumoniae infection . The increase in bacterial burden did not appear to be due to differences in the numbers of neutrophils ( CD11b+ Ly6Ghi ) because neutrophils were detected at comparable levels in WT and Skap2-/- lungs , bone marrow as well as bronchoalveolar lavage of infected mice ( Figure 1C; Figure 1—figure supplement 1A–D ) . In addition , there was no difference in alveolar macrophages ( CD11bint CD11chi ) , resident monocytes ( CD11b+ Gr1lo ) , or dendritic cells ( CD11bhi CD11chi ) in the lungs of K . pneumoniae-infected WT and Skap2-/- mice ( Figure 1C; Figure 1—figure supplement 1E–J ) . Histological examination showed extensive leukocyte infiltration in both the WT and Skap2-/- infected lungs supporting the flow cytometry data ( Figure 1D–L , black arrows ) . This interstitial infiltration inflammatory cells can be seen in the early stages of infection prior to bronchopneumonia ( Lawlor et al . , 2005; Xiong et al . , 2015 ) . Furthermore , some infected Skap2-/- lungs ( 2/5 lungs ) had lesions with cellular destruction and uncontrolled bacterial growth surrounded by sparse leukocyte infiltration ( Figure 1K–L , blue arrows ) . To evaluate whether SKAP2-dependent control of K . pneumoniae colonization was required in a different mouse background , C57BL/6J , C57BL/6J Skap2-/- , or heterozygous littermates were inoculated retropharyngeally with K . pneumoniae ( Figure 1M–N; Figure 1—figure supplement 1K–N ) . At 24 hpi , C57BL/6 Skap2-/- mice had a significant increase in bacterial burden compared to C57BL/6 and heterozygous littermates ( Figure 1M ) . In general , comparable numbers of neutrophils and inflammatory monocytes were detected in all three strains ( Figure 1N; Figure 1—figure supplement 1L–N ) although the percent of neutrophils recovered in C57Bl/6 Skap2-/- mice was higher ( Figure 1N ) In summary , although leukocytes are present in the Skap2-/- lungs at comparable or slightly higher level to wild-type lungs , Skap2-/- mice were more susceptible to K . pneumoniae infection suggesting that their antimicrobial functions are compromised . To investigate whether Skap2 expression in bone marrow-derived leukocytes was required for controlling the antibacterial response to K . pneumoniae infection , we generated bone marrow ( BM ) chimeric mice by transplanting WT or Skap2-/- BM cells into either WT or Skap2-/- mice ( Figure 2A ) . Transplantation efficiency was confirmed by flow cytometry ( Figure 2—figure supplement 1 ) . WT recipient mice reconstituted with Skap2-/- BM had significantly more K . pneumoniae in contrast to WT mice transplanted with WT BM ( Figure 2B ) . Conversely , Skap2-/- recipients with WT BM cells had significantly decreased K . pneumoniae in their lungs as compared to Skap2-/- hosts that received Skap2-/- BM cells ( Figure 2B ) . The differences in bacterial burdens were not due to differences in numbers of neutrophils as equivalent numbers were recovered among all four groups of mice ( Figure 2C; Figure 2—figure supplement 1C–E ) . While fewer inflammatory monocytes ( iMOs ) were detected in Skap2-/- recipients regardless of BM cell donor , the difference did not correlate with bacterial load ( Figure 2B , D; Figure 2—figure supplement 1F–H ) . Combined , these data indicate Skap2 expression in BM-derived immune cells is required to contain K . pneumoniae growth during lung infection , and that Skap2 expression in cells that are not derived from the marrow do not contribute to this phenotype . The contribution of neutrophils and iMOs to host defense against K . pneumoniae infection is dependent on the bacterial strain used with K . pneumoniae ATCC 43816 being more susceptible to neutrophils ( Xiong et al . , 2015; Silver et al . , 2019 ) . To determine the role of neutrophils and iMOs in the increased susceptibility of Skap2-/- mice to K . pneumoniae , WT and Skap2-/- mice were treated with the α-Ly6G ( clone 1A8 ) to deplete neutrophils , or the MC-21 antibody , which targets CCR2+ cells to deplete iMOs ( Figure 2E ) . Depletion efficiency was confirmed by flow cytometry ( Figure 2—figure supplement 2A–F ) . While the K . pneumoniae burden in iMOs-depleted WT mice was similar to vehicle-treated mice , significantly more bacteria were recovered from the lungs of neutrophil-depleted WT mice ( Figure 2E ) confirming the importance of neutrophils in host defense ( Ye et al . , 2001; Xiong et al . , 2015 ) . The neutrophil-depleted WT mice had similarly high bacterial burdens as Skap2-/- mice ( Figure 2E ) , and the depletion of Skap2-/- mice of neutrophils or iMOs did not increase their bacterial burden suggesting that protection against K . pneumoniae infection is SKAP2-dependent ( Figure 2E ) . To assess the contribution of Skap2 expression in BM-derived iMOs and neutrophils in limiting K . pneumoniae infection , iMOs and neutrophils were depleted from irradiated Skap2-/- mice that had been reconstituted with WT BM cells ( Figure 2F ) . Depletion efficiency was confirmed by flow cytometry ( Figure 2—figure supplement 2G–J ) . As expected , reconstitution of Skap2-/- mice with WT BM resulted in a significant reduction in bacterial burden compared to Skap2-/- mice that received Skap2-/- BM cells ( Figure 2F ) . Skap2-/- mice that received WT BM and were then depleted of neutrophils with α-Ly6G showed an increase in bacterial load and that burden was similar to those in Skap2-/- mice that received Skap2-/- neutrophils ( Figure 2F ) . By contrast , α-CCR2-treated mice had a similar bacterial burden as PBS-treated mice ( Figure 2F ) . Together , these data indicate that neutrophils cannot control K . pneumoniae 43816 infection in the absence of SKAP2 in the hematopoietic compartment . As Skap2-/- neutrophils are present in infected tissues ( Figure 1D , J , N; Figure 1—figure supplement 1A–D ) , the uncontrolled K . pneumoniae growth may occur because of defects in the antimicrobial functions of Skap2-/- neutrophils at the site of infection . Neutrophils have multiple antimicrobial mechanisms to limit bacterial growth during infection ( Futosi et al . , 2013; Mócsai , 2013 ) . To investigate which antimicrobial functions are controlled by SKAP2 , the ER-Hoxb8 conditionally-immortalized stem cell system was adopted as a tool to produce a supply of neutrophils ( Wang et al . , 2006; Chu et al . , 2019 ) . Mouse BM-derived stem cells from WT and Skap2-/- mice were retrovirally transduced with Hoxb8 regulated by estrogen ( ER-Hoxb8 ) , and maintained in media containing estrogen ( E2 ) and stem cell factor ( SCF ) to expand granulocyte-monocyte progenitors ( GMP ) in vitro ( Figure 3—figure supplement 1A–I and see Materials and Methods ) . Removal of E2 inactivates Hoxb8 expression and the addition of interleukin-3 and granulocyte colony-stimulating factor led to terminal neutrophil differentiation ( Figure 3—figure supplement 1A–I ) . Neutrophil maturation from WT and Skap2-/- ER-HoxB8-immortalized GMP cells ( Hoxb8 GMP ) was evaluated by nuclear morphology ( Figure 3—figure supplement 1B ) and cell surface expression of cKit , CD11b and Ly6G ( Figure 3—figure supplement 1C–D , G–I ) . Hoxb8 GMP cells were mononuclear and cKit+ CD11b- Ly6G- ( Figure 3—figure supplement 1C–D , G–I ) . Cells from both mouse lines differentiated by day 4 and were comparably viable ( Figure 3—figure supplement 1E–F ) . Fully differentiated cells are referred to as differentiated in vitro ( DIV ) neutrophils . Like BM-derived neutrophils , DIV neutrophils expressed polymorphonuclear morphology and are cKit- CD11b+ Ly6G+ ( Figure 3—figure supplement 1B–D , G–I ) . To confirm the functionality of DIV neutrophils in comparison to BM neutrophils , their ability to generate reactive oxygen species ( ROS ) following integrin and Fcγ receptor stimulation was assessed ( Chu et al . , 2019 ) . When plated onto surfaces coated with poly-RGD , an engineered polypeptide containing multiple copies of the integrin binding motif , WT BM and DIV neutrophils released superoxide , while Skap2-/- BM and DIV neutrophils did not ( Figure 3A–C ) . This is consistent with previously published data in C57BL/6J and Skap2-/- BM neutrophils ( Boras et al . , 2017 ) . The loss in integrin-mediated ROS production was not due to defects in the NADPH oxidase itself , as Skap2-/- BM and DIV neutrophils robustly released ROS when stimulated with phorbol myristate acetate ( PMA ) , which bypasses receptor-mediated signaling pathways and activates NADPH oxidase via protein kinase C ( PKC ) ( Dang et al . , 2001; Figure 3C ) . By contrast , after stimulation of Fcγ receptors by IgG immune complexes ( IC ) , WT and Skap2-/- BM and DIV neutrophils robustly produced ROS ( Figure 3D–F ) . Skap2-/- BM and DIV neutrophils consistently released 60–80% as much ROS as compared to levels seen in comparable WT neutrophils . This indicates IC-stimulated ROS is partially dependent on SKAP2 . Differences in ROS production were not due to differences in surface marker expression levels as WT and Skap2-/- DIV neutrophils expressed similar levels of integrin and Fcγ receptors as measured by CD11b and CD16 expression , respectively , by flow cytometry ( Figure 3G–J ) . Combined , these results indicate WT and Skap2-/- DIVs phenocopy primary BM neutrophils both morphologically and functionally and are a useful tool to investigate how SKAP2 modulates K . pneumoniae-induced neutrophil responses . Furthermore , these data suggest that a functional NADPH complex can assemble in Skap2-/- neutrophils and that SKAP2 is required for receptor-activated signaling pathways downstream of integrin receptors , but not Fcγ receptors . Multiple neutrophil antimicrobial mechanisms , including degranulation , phagocytosis , and NETs , have been implicated in host defense against K . pneumoniae infection ( Chen et al . , 2016; Ivin et al . , 2017; Papayannopoulos et al . , 2010; Bengoechea and Sa Pessoa , 2019; Paczosa and Mecsas , 2016; Hirche et al . , 2005; Zhao et al . , 2015; Kobayashi et al . , 2016 ) . To examine whether Skap2-/- neutrophils degranulated as efficiently as WT neutrophils during K . pneumoniae infection , we measured levels of released MMP-9 and neutrophil elastase ( ELA2 ) , proteins found in tertiary and primary granules , respectively ( Papayannopoulos et al . , 2010; Sheshachalam et al . , 2014 ) . Supernatants from WT and Skap2-/- K . pneumoniae-infected lungs had equivalent levels of MMP-9 and ELA2 in Skap2-/- at 16 and 24 hpi as detected by ELISA ( Figure 4A–B ) . This suggests that the increased in susceptibility of Skap2-/- mice was not due to defects in degranulation . To determine the role of SKAP2 in phagocytosis of K . pneumoniae , phagocytosis was initially measured using microscopy . However , microscopic examination of WT DIV neutrophils infected with GFP-labeled K . pneumoniae and counterstained with Alexa594-conjugated polyvalent α-K . pneumoniae for extracellular bacteria did not reveal any internalized bacteria indicating that phagocytosis levels were less than 1% ( unpublished data ) . Using a more sensitive gentamicin protection assay ( Walker et al . , 2019 ) , K . pneumoniae 43816 was recovered on average at less than 0 . 01% in WT and Skap2-/- DIV neutrophils , while a non-encapsulated ( ΔcpsB ) K . pneumoniae strain was recovered at approximately 0 . 05% ( Figure 4C ) . This low level was not due to a defect in phagocytosis of DIV neutrophils as WT and Skap2-/- DIV neutrophils were able to phagocytose Yersinia pseudotuberculosis YPIII ΔyscNU strain , a strain that is susceptible to phagocytosis ( Balada-Llasat and Mecsas , 2006 ) . WT and Skap2-/- DIV neutrophils phagocytosed 20% and 10% of ΔyscNU , respectively ( Figure 4C ) , suggesting that phagocytosis of this Y . pseudotuberculosis strain is partially dependent on SKAP2 . Although we observed low level of phagocytosis of K . pneumoniae 43816 , the SKAP2-mediated defect in phagocytosis might be more prominent when challenged with other K . pneumoniae isolates . Combined , these results indicate that the K . pneumoniae 43816 strain is resistant to neutrophil phagocytosis regardless of SKAP2 expression , but can elicit SKAP2-independent degranulation during infection . ROS is an important mechanism for protection against K . pneumoniae as mice that lack the essential gp91phox component of NADPH oxidase , Cybb-/- , were significantly more susceptible to K . pneumoniae infection and had a significant increase in bacterial burden in their lungs ( Figure 5—figure supplement 1A; Paczosa et al . , 2020 ) . To test whether SKAP2 deficiency resulted in a defect in neutrophil ROS production after K . pneumoniae infection , an isoluminol-amplified chemiluminiscence assay was used . In contrast to WT BM and DIV neutrophils , neither Skap2-/- BM nor DIV neutrophils produced ROS following exposure to K . pneumoniae ( Figure 5A–B; Figure 5—figure supplement 1B-C ) . The loss of K . pneumoniae-stimulated ROS production was not due to a defect in bacterial-binding as WT and Skap2-/- DIV neutrophils bound GFP-labeled K . pneumoniae equivalently when assessed by flow cytometry following both 15- and 30 min incubations ( Figure 5C ) . Without treating the cells with actin inhibitor , such as cytochalasin B , bound and phagocytosed bacteria could contribute to the levels of GFP+ cells . However , the less than 1% of internalization of K . pneumoniae observed in the gentamicin protection assay ( Figure 4C ) suggests that the majority of the bacteria are extracellular , and that GFP+ cells are more likely reflective of binding . Finally , SKAP2 was important for ROS production after infection with another gram-negative bacterial strain , a type 3 deficient Y . pseudotuberculosis strain , ΔyscF ( Figure 5—figure supplement 1D ) . In summary , Skap2-/- neutrophils are defective in triggering the signaling cascade ( s ) required for ROS after K . pneumoniae binding to surface receptors . To investigate how SKAP2 mediates K . pneumoniae-induced ROS production , we first evaluated the requirement for several well-established proteins that are critical for receptor-mediated ROS production after K . pneumoniae infection . Src family kinases ( SFKs ) , Spleen Associated Tyrosine Kinase ( Syk ) , Bruton’s tyrosine kinase ( Btk ) , Phospholipase Cγ2 ( PLCγ2 ) , and PKC play critical roles in receptor-mediated ROS production ( Löfgren et al . , 1999; Braselmann et al . , 2006; Volmering et al . , 2016; Dang et al . , 2001; Raad et al . , 2009; Kovács et al . , 2014; Fumagalli et al . , 2007; Graham et al . , 2007; Nguyen et al . , 2017 ) . To determine whether these proteins are required for K . pneumoniae-stimulated ROS production , WT DIV neutrophils were pretreated with PP2 , R406 , Ibrutinib , U-73122 , and Go6983 , which are small molecules that inhibit the functions of SFKs , Syk , Btk , PLCγ2 , and PKC ( both novel and conventional isoforms ) , respectively ( Figure 5D; Peterman et al . , 2004; Braselmann et al . , 2006; Rolán et al . , 2013; Prezzo et al . , 2019; Fumagalli et al . , 2007 ) . The DIV neutrophils were then infected with K . pneumoniae . Pretreatment with PP2 , R406 , Ibrutinib , U-73122 , and Go6983 abolished ROS production after K . pneumoniae infection compared to vehicle-pretreated neutrophils ( Figure 5E ) . As expected , only pretreatment with Go6983 blocked ROS after PMA exposure . U73433 , an U73122 analog that does not target PLCγ , did not block K . pneumoniae- and PMA-induced ROS ( Figure 5E; Rolán et al . , 2013 ) . This suggests SFKs , Syk , Btk , PLCγ2 , and PKC are each necessary for K . pneumoniae-activated ROS production . Next , we assessed whether SKAP2 mediates K . pneumoniae-induced ROS production by influencing tyrosine phosphorylated-dependent functions of SFKs and Syk . To determine whether SFKs and Syk are phosphorylated in response to K . pneumoniae infection , western blots of lysates from K . pneumoniae-infected WT DIV neutrophils were probed with phospho-specific antibodies . Infection with K . pneumoniae induced phosphorylation of SFKs ( pSFK ) and Syk ( pSFK ) to levels 2–5-fold higher than in unstimulated neutrophils ( Figure 5F–I; Supplementary file 1 ) . These results support findings with inhibitors showing that SFKs and Syk functions are required for K . pneumoniae-activated ROS ( Figure 5E ) . In addition , proline-rich tyrosine kinase 2 ( Pyk2 ) , which is a tyrosine kinase that functions downstream of SFKs and Syk to mediate ROS production ( Kamen et al . , 2011; Zhao and Bokoch , 2005 ) , was phosphorylated in response to K . pneumoniae stimulation ( Figure 5K–L; Supplementary file 1 ) . Thus , signaling circuit ( s ) containing SFKs , Syk , and Pyk2 were activated in neutrophils following receptor binding of K . pneumoniae . To determine if SKAP2 is required for phosphorylation of these tyrosine kinases , the levels of pSFKs , pSyk , and pPyk2 were assessed in Skap2-/- DIV neutrophils following K . pneumoniae exposure . While SFKs , Syk , and Pyk2 phosphorylation were all slightly induced in Skap2-/- neutrophils , the level of induction was often significantly reduced as compared to WT neutrophils ( Figure 5F–L; Supplementary file 1 ) . To determine if the reduction in phosphorylation was due to decreased cell viability , Skap2-/- DIV neutrophils were also stimulated with IC as a control . Stimulation of FcγRs resulted in the phosphorylation for SFKs , Syk , and Pyk2 to similar levels in both WT and Skap2-/- DIV neutrophils ( Figure 5F , H , K and Supplementary file 1 ) . Collectively , these results show that SFKs , Syk , and Pyk2 are required for K . pneumoniae-stimulated ROS production , and that the loss of SKAP2 prevents full phosphorylation of SFKs , Syk , and Pyk2 after K . pneumoniae stimulation such that neutrophils are unable to generate ROS production .
Our experiments reveal that SKAP2 plays a key role in host defense in the hematopoietic compartment against K . pneumoniae 43816 in pulmonary infections in a murine lung model of infection . SKAP2 was indispensable for K . pneumoniae-stimulated neutrophil ROS production , but not degranulation or phagocytosis , suggesting that the increased bacterial burden in the Skap2-/- mice is affected by the loss of ROS . Sensitivity of K . pneumoniae to ROS is strongly supported by the increased bacterial burden in Cybb-/- mice during lung infection , and observations in CGD patients ( Wolach et al . , 2017; Paczosa et al . , 2020 ) . In murine neutrophils , SKAP2 was required for full phosphorylation of SFKs , Syk , and Pyk2 , demonstrating a critical role in K . pneumoniae-induced signaling pathways that are required for ROS production . Consistent with previous findings ( Regueiro et al . , 2006; March et al . , 2013 ) , K . pneumoniae 43816 was resistant to internalization indicating that phagocytosis is most likely not the primary protective mechanism provided by neutrophils to this strain . Prior studies , including one using K . pneumoniae 43816 , have shown that degranulation of myeloperoxidase , ELA2 , and cathepsin G are important for reducing bacterial burden of K . pneumoniae in mice ( Hirche et al . , 2005; Zhao et al . , 2015 ) . However , we found that despite equivalent levels of ELA2 , degranulation in Skap2-/- mice was not sufficient to control K . pneumoniae burden in the absence of ROS , indicating that neutrophil ROS and degranulation may cooperate in containing K . pneumoniae infection . Since degranulation occurred normally in Skap2-/- lungs , the pathways of ROS production are distinct from signaling pathways regulating degranulation in murine neutrophils after K . pneumoniae infection . Neutrophils and ROS contribute to the defense against other pathogens such as Y . pseudotuberculosis , Staphylococcus aureus , Candida albicans , among others ( Green et al . , 2016; Mócsai , 2013; Nguyen et al . , 2017 ) . The immunological mechanisms of SKAP2-mediated neutrophil responses to other pathogens are under investigation . We propose the following model for SKAP2-dependent neutrophilic response to K . pneumoniae in mice based our findings and work of others ( Figure 6; Alenghat et al . , 2012; Swanson et al . , 2008; Togni et al . , 2005; Timms et al . , 1999 ) . Following K . pneumoniae infection , activation of receptors , like GPCRs recognizing N-formylated bacterial proteins or mincle recognizing bacterial lectins , leads to the activation of phosphoinositide 3 kinase ( PI3K ) and the generation of phosphatidylinositol [3 , 4 , 5]-triphosphate ( PIP3 ) , resulting in PIP3-mediated SKAP2 activation and membrane localization ( Figure 6A–B; Futosi et al . , 2013; Swanson et al . , 2008; Sharma et al . , 2014; Alenghat et al . , 2012 ) . The activated ( open ) phosphorylated SKAP2 complex can then bind to and enhance phosphorylation of SFKs shifting their active/inactive equilibrium to more active forms and leading to Syk activation ( Figure 6B; Marie-Cardine et al . , 1998; Bureau et al . , 2018; Kouroku et al . , 1998; Lowell , 2011 ) . This is supported by previous biochemical and pull-down studies showing that SKAP2 can directly interact with SFKs ( Bureau et al . , 2018; Liu et al . , 1998; Wang and Rudd , 2008 ) . SFKs and Syk may directly associate with and activate Pyk2 , as observed in response to GPCRs , integrin , TNFα , and/or Salmonella stimulation ( Dikic et al . , 1996; Evangelista et al . , 2007; Han et al . , 2003; Mócsai et al . , 2002 ) . Furthermore , SKAP2 may interact with Pyk2 signaling complex directly or indirectly through their association with Sirpα ( Raab et al . , 2017; Alenghat et al . , 2012 ) . Any of these mechanisms would promote stabilization and propagation of SFKs/Syk/Pyk2 phosphorylation in activated murine neutrophil leading to ROS . While SFKs , Syk , and Pyk2 have been shown to be important for degranulation in murine and human neutrophils ( Van Ziffle and Lowell , 2009; Kamen et al . , 2011; Mócsai et al . , 1999; Han et al . , 2003 ) , we observed degranulation in murine lungs and low levels of phosphorylation after K . pneumoniae infection in murine neutrophils in vitro . These low levels may be sufficient to induced degranulation through a SKAP2-independent signaling pathway . Alternatively , specific phosphorylation sites of SFKs or Syk not tested here may still occurred in the absence of SKAP2 and be sufficient to induce degranulation ( Carsetti et al . , 2009; Tsang et al . , 2008; Moroco et al . , 2014; Mócsai et al . , 2010 ) . In addition , different domains of Pyk2 regulate ROS and degranulation ( Han et al . , 2003 ) . Specifically , the inhibition of the carboxy-terminus of Pyk2 in human neutrophils leads to a reduction in ROS production in response to TNF , Salmonella , and Listeria , but did not impair neutrophil degranulation and bacterial killing ( Han et al . , 2003 ) . SKAP2 may impact the carboxy-terminus of Pyk2 in murine neutrophils , but not activation of other domains . Skap2-/- mice have normal levels of circulating neutrophils and Skap2-/- neutrophils were present in K . pneumoniae-infected lungs at a similar , and sometimes higher , levels as WT neutrophils ( Figure 1C–D; Figure 1—figure supplement 1A ) and ( Togni et al . , 2005 ) . However , prior studies revealed that Skap2-/- myeloid cells , including neutrophils , have impaired recruitment and migration into tissues during sterile inflammation ( Tanaka et al . , 2016; Boras et al . , 2017 ) . This suggests that migration to sterile , but not infectious sites , may require SKAP2 , which is consistent with findings with Syk . Syk is required for neutrophil migration in an inflammatory model but not during bacterial infection ( Zarbock et al . , 2007; Frommhold et al . , 2007; Van Ziffle and Lowell , 2009; Schymeinsky et al . , 2006 ) . Alternatively , it is possible that Skap2-/- neutrophils may have a reduced migration level in K . pneumoniae-infected lungs but this is balanced by significantly higher rate of neutrophil survival and these two factors result in a net neutrophil level that is equivalent to or higher than that observed in WT mice . This result could arise because ROS production can increase myeloid cell death ( Coxon et al . , 1996; Geering and Simon , 2011; Geering et al . , 2011 ) , and thus , defects in ROS production by Skap2-/- neutrophils could contribute to higher survival and accumulation in infected murine lungs . These possibilities are important to address with additional experiments , such as pulse-labelling newly differentiated neutrophils to track rates of migration during infection in vivo . Murine models of infection have been helpful in illuminating K . pneumoniae infection biology , however , there exist significant differences between mice and humans in the immune response to infection , including disparities in structural anatomy , circulating neutrophil counts , surface receptors , and signaling cascades , among others ( Mizgerd and Skerrett , 2008; Mestas and Hughes , 2004; Condliffe et al . , 2005; Hajjar et al . , 2010; Sun et al . , 2016; Bagaitkar et al . , 2012; Bengoechea and Sa Pessoa , 2019 ) . Thus , to date it is unknown if SKAP2 plays a role in mediating the human neutrophil response to K . pneumoniae infection . Skap2 has 9 transcripts and at least 50 exonic variants that have been reported in the human population , however , their functional consequences , if any , remains unclear ( Cunningham et al . , 2019 ) . It would be interesting to explore if the SKAP2-mediated K . pneumoniae-stimulated responses occur in human neutrophils , and whether neutrophils ( or other cell types ) from individuals carrying specific Skap2 variants respond differently K . pneumoniae 43816 infection . In addition , it is unclear whether our results with K . pneumoniae 43816 will be found with other K2 strains or strains of other serotypes . Genome-wide analyses of K . pneumoniae clinical isolates have shown extensive genetic diversity , virulence factors , and pathogenicity ( Holt et al . , 2015; Deleo et al . , 2014; Agard et al . , 2019 ) . Furthermore , since neutrophils are not the major player in host defenses against all strains of K . pneumoniae , including some multi-drug resistant clinical isolates ( Xiong et al . , 2016; Xiong et al . , 2015; Broug-Holub et al . , 1997; Greenberger et al . , 1996 ) , future studies can use these K . pneumoniae strains to probe the role of SKAP2 in neutrophils as well as other hematopoietic cells during K . pneumoniae infections . In summary , neutrophils and their production of ROS play a dual role in host health in many disease states from acute infections to autoimmune diseases to cancer ( Hoffmann and Griffiths , 2018; Nguyen et al . , 2017 ) , and the crucial function of SKAP2-mediated ROS in murine neutrophils suggests that further analysis of the role of SKAP2 in human health warrants investigation .
BALB/c and C57BL/6J mice were purchased from Taconic Biosciences and Jackson laboratory ( Bar Harbor , ME ) , respectively . Mating pairs of Cybb-/- in C57BL/6J background , and Skap2+/- ( B6 . 129S5-Skap2Gt ( VICTR20 ) 21Lex/Mmjax ) were purchased from Jackson laboratory and bred at Tufts University in the specific pathogen-free facility of Tufts University . Generation of Skap2-/- mice in the BALB/c background was previously described ( Togni et al . , 2005; Alenghat et al . , 2012 ) . Skap2-/- mice were genotyped by two independent PCRs using primer sequences: SkapLeft-forward primer ( common for both PCRs ) , 5’ CAG CTT GCC GAC TTT TCT; GTLexVir , 5’GAG GGC TGG ACC GCA TCT GG; GTSkapRight , 5’CCG CCT CCC ACC CCT CAA TC following procedures described from Jackson laboratory and ( Alenghat et al . , 2012 ) . All mice were handled in accordance with protocols approved by the Institutional Animal Care and Use Committee of Tufts University . Mouse infections were carried out as previously described ( Paczosa et al . , 2020; Silver et al . , 2019 ) with the following modifications . MKP220 , streptomycin-resistant derivative of K . pneumoniae ATCC43816 strain was used for all infections ( Paczosa et al . , 2020; Silver et al . , 2019 ) . Isoflurane anesthetized mice on a BALB/c background were intranasally infected dropwise with 5 × 103 cfu K . pneumoniae in 40 μl of sterile PBS , or with PBS ( Fisher Scientific ) alone . C57BL/6J mice were inoculated retropharyngeally with 5 × 103 cfu as previously described ( Paczosa et al . , 2020 ) . This change in infection route was because we have found that if delivered intranasally , C57BL/6J mice will rapidly dispel the inoculum through their nose and mouth resulting in inconsistent infection . Mice were euthanized using CO2 asphyxiation at the indicated time points after infection . Lungs were collected into sterile PBS , weighed , and aseptically homogenized by pushing the tissue through a 70 μM cell strainer . For bacterial burden analyses , serially diluted homogenates were plated on L-agar and incubated at 37°C overnight to determine CFU/g lung . For depletion of neutrophils or iMOs , mice were intraperitoneally injecting 100 μl of 50 μg/ml α-Ly6G antibody ( clone: 1A8 , Fisher Scientific ) , or 100 μl of 200 μg/ml α-MC21 antibody 16 hr pre-infection . Depletion efficiency was measured by flow cytometry as described below . Collection of bronchoalveolar lavage was conducted as previously described ( Silver et al . , 2019 ) . 4–5 week-old BALB/c and Skap2-/- mice received 100 mg/kg/24 hr SulfaTrim available ad libitum in drinking water for a week prior to and a week following whole body irradiation . Mice were lethally irradiated with 2 doses of 4 . 5 Gγ with a 3–4 hr interval between doses . The next day , each mouse intravenously received 100 μl of sterile PBS containing 5 × 106 BM cells from either BALB/c or Skap2-/- donor mice ( 7–15 week-old ) and were infected with K . pneumoniae 4–5 weeks later . For histological studies , lungs from mock or K . pneumoniae-infected mice were perfused through a tracheal incision using 4% formaldehyde ( MP Biomedicals ) , removed , and incubated in fresh 4% formaldehyde overnight at room temperature ( RT ) . Excess fatty tissues were removed , and lungs were placed in histology cassettes for processing by the Tufts Animal Pathology Core . Tissues were embedded in paraffin and 7 μM thick sections were sliced and stained with hematoxylin and eosin ( HE ) . Sections were blindly scored for number of areas of leukocyte infiltration and detectable bacteria by 4–7 individuals with at least 3 lung sections per mouse using light microscopy . Three PBS-inoculated and 5 K . pneumoniae-infected mice were scored per genotype . Tissues were imaged using an Echo Revolve R4 microscope in the upright brightfield configuration at 4x ( NA 0 . 13 ) or 40x ( NA 0 . 75 ) magnification and acquired at RT using a 12 MP CMOS color camera and the Echo Revolve built-in software . After passage of lung tissues through 70 μm strainers , the cell suspensions were centrifuged at 13 , 000 x g for 10 min at 4°C . Supernatants were then transferred into new Eppendorf tubes for measurement of release of matrix metalloproteinase ( MMP-9 ) , and neutrophil elastase ( ELA-2 ) by DuoSet ELISA kits ( R and D Systems ) per manufacturer’s protocols . After passage of lung tissues through a 70 μm strainer , cell suspensions were treated with 1 mg/ml Collagenase D ( ThermoFisher Scientific ) for 1 hr at 37°C , and then with 1X Pharm Lyse ( Fisher Scientific ) for 5 min at 4°C . Cells were resuspended in ice-cold fluorescence-activated cell sorter ( FACS ) buffer ( PBS containing 1% FBS ) , and blocked with rat anti-mouse CD16/CD32 ( Mouse BD Fc Block BD Biosciences ) for 10 min at 4°C . For time courses , cells were stained with α-CD11b-PE ( BioLegend ) , α-Ly6G PE-Cy7 ( BioLegend ) , α-CD11c-PerCP-Cy5 . 5 ( BD Biosciences ) , and α-Gr1-FITC ( BioLegend ) for analysis of neutrophils ( CD11b+ Ly6G+ ) , alveolar macrophages ( CD11bint CD11chi ) , resident monocytes ( CD11b+ Gr1lo ) , and dendritic cells ( CD11bhi CD11chi ) . For bone marrow chimeric and depletion experiments , lung cells were stained with α-CD11b-PacBlue ( BioLegend ) , α-Ly6G-PE-Cy7 ( BioLegend ) , α-Gr1-APC ( BioLegend ) , and α-Ly6C-AlexaFluor647 ( BioLegend ) for analysis of neutrophils and inflammatory monocytes as indicated in figure legends . For some experiments , cells were fixed with 4% formaldehyde , permeabilized with 0 . 1% saponin , and intracellularly stained with α-SKAP2 ( Proteintech ) or rabbit IgG polyclonal isotype antibody ( Proteintech ) , followed by goat anti-rabbit AlexaFluor488 secondary antibody ( ThermoFisher Scientific ) . Data were collected by a BD LSRII , and analyzed with FlowJo ( version 10 . 1 ) . Mouse BM neutrophils were isolated as previously described ( Rolán et al . , 2013 ) with the following modifications . Neutrophils were isolated using a three step Percoll density gradient ( 55% , 65% , and 75% ) , and centrifuged at 480 x g for 30 min at 26°C without applying the break . Neutrophils were collected at the 65–75% interface , resuspended in HBSS without Ca2+ or Mg2+ ( HBSS- ) , then centrifuged at 250 x g for 5 min at RT to wash and pellet the cells . Supernatants were removed and cells were resuspended in HBSS- and centrifuged for a total of three washes . Neutrophils were rested at RT for one hour , resuspended in HBSS with Ca2+ and Mg2+ ( HBSS+ ) to the desired concentration , incubated at RT for 20 min , and then shifted to 37°C for 10 min prior to the experiment . The purity of neutrophil preparations was verified by staining an aliquot of the cells with α-CD11b-PacBlu , α-Ly6G-PE-Cy7 , and α-Gr1-APC and analyzed by flow cytometry . Immortalization of murine BM cells with ER-HoxB8 was done as previously described ( Wang et al . , 2006 ) with the following modifications . Bone marrow stem cells were collected from the tibias and femurs of mice and passed through a 40 μm filter . Cells were layered over Ficoll-Paque-Plus ( Pharmacia ) , and centrifuged at 400 x g for 25 min at RT without applying the break to enrich for mononuclear cells . These cells were incubated in a 6-well tissue culture plate ( Greiner Bio-One ) for 24 hr at 37°C with 5% CO2 prior to the retroviral transduction in complete RPMI , cRPMI ( RPMI-1640 with 10% FBS , 2 mM L-glutamine , and 100U penicillin and 0 . 1 mg/ml streptomycin ) , supplemented with 10 ng/ml stem cell factor ( SCF ) , 10 ng/ml interleukin-3 ( IL-3 ) , and 10 ng/ml interleukin-6 ( IL-6 ) . The next day , non-adherent cells were harvested and 5 × 105 cells in 500 μl of cRPMI were plated onto a 12-well tissue culture plate ( Corning ) coated with 10 μg/ml human fibronectin ( Sigma ) . Retroviral supernatant ( MSCVneo-HA-ER-Hoxb8 ) was applied in the presence of 8 μg/ml polybrene and the plates were centrifuged for ‘spinoculation’ at 1000 x g for 90 min at RT . Following spin infection , cells were maintained in cRPMI supplemented with 0 . 5 μM beta-estradiol ( Sigma , E2 ) and conditioned media containing approximately 50 ng/ml SCF ( SCF + E2 cRPMI ) . Cells were maintained for 3–4 weeks to select for ER-HoxB8-carrying granulocyte-monocyte progenitor cells . Uninfected cells were maintained alongside as controls and all died by 4 weeks . To confirm the identify of cell lines , we assessed for SKAP2 protein expression by intracellular staining or western blotting . Mycoplasma testing of cultured lines was conducted at 3–6 month intervals using e-Myco Mycoplasma PCR Detection Kit ( Bulldog Bio ) . ER-HoxB8 stem cell lines were grown in E2 + SCF cRPMI . Differentiation into neutrophils was done as previously described ( Wang et al . , 2006; Pelletier et al . , 2017 ) with the following modifications . Aliquots of ER-HoxB8 progenitors were washed 3 times in PBS and cultured in E2-free cRPMI media supplemented with ~50 ng/ml SCF , IL-3 , and GCSF for two days . Cells were then restimulated with 50 ng/ml GCSF for another 48 hr . At this point , cells were differentiated and termed DIV neutrophils . DIV neutrophils were collected , pelleted at 250 x g for 5 min at 4°C , re-suspended in HBSS- and rested for 1 hr at RT . DIV neutrophils were re-suspended in HBSS+ to the desired concentration , incubated at RT for 20 min , and then shifted to 37°C for 10 min for functional assays . DAPI staining and flow cytometry were performed to confirm that >90% of cells were neutrophils . An aliquot of 1 × 105 BM and DIV neutrophils were spun onto microscope slide using Shannon Cytospin 3 , fixed with 4% formaldehyde , stained with DAPI ( ThermoFisher Scientific ) , and mounted using ProLong Gold antifade reagent ( Invitrogen ) . Cell morphology was imaged at 60X by an Echo Revolve R4 microscope in the upright fluorescent configuration . Aliquots of cells were resuspended in cold FACS buffer and blocked with Mouse BD Fc Block ( BioLegend ) for 10 min at 4°C . Cells were then stained with α-CD11b-PacBlu , α-Ly6G-PE-Cy7 , and α-cKit-PE , and then fixed with 4% formaldehyde prior to analysis . Viability was assessed using trypan blue ( Gibco ) exclusion test immediately prior to functional studies to confirm that >85% of cells were live and all counts were based on live cells . In addition , an ATP-based assay , Promega CellTiter-Glo Luminescent Cell viability Assay , was conducted during the time course of several experiments to assess viability per instructor’s instructions ( Promega ) ( Figure 1—figure supplement 1E-F ) . Neutrophils ( 1 × 105 cells/well ) and green fluorescent protein ( GFP ) -labeled K . pneumoniae ( MOI 40 ) were added to non-tissue-treated 96-well plates ( CellTreat ) , centrifuged at 250 x g for 3 min , and then incubated for 30 min at 37°C with 5% CO2 for 15 or 30 min . Cells were then harvested , centrifuged at 250 x g for 5 min , and resuspended in 200 ul/well ice-cold FACS buffer; this was repeated for a total of 3 washes . Cells were then fixed with 4% formaldehyde , and nuclei were stained with DAPI . The percentage of K . pneumoniae-associated neutrophils was determined by flow cytometry using uninfected neutrophils as control . K . pneumoniae MKP220 and a isogenic non-encapsulated ΔcpsB strain ( Paczosa et al . , 2020; Silver et al . , 2019 ) were grown overnight in LB media at 37°C with aeration , and the next day diluted 1:40 into fresh LB media and grown for an additional 2 hr at 37°C with aeration . The Y . pseudotuberculosis YPIII ΔyscNU strain ( Balada-Llasat and Mecsas , 2006 ) was grown in 2XYT at 26°C with aeration . K . pneumoniae and Y . pseudotuberculosis were then used to infect 1 × 105 DIV neutrophils at an MOI of 10 for 30 min at 37°C in the presence of 5% CO2 in 96-well 4HBX plates pre-coated with 10% FBS , and then 100 μg/ml gentamicin was added to wells for 1 hr at 37°C to kill extracellular bacteria . Control FBS-coated wells contained bacteria but no neutrophils and were not treated with gentamicin ( non-gentamicin-treated wells ) . All following steps were conducted at 4°C . Cold PBS was added to the wells; plates were centrifuged at 250 x g for 5 min , and all media were gently removed; this was repeated for a total of four washes . All wells were treated with 0 . 1% Triton X-100 ( Sigma-Aldrich ) for 10 min at 4°C , and then serially diluted and plated to quantify surviving bacteria . The percent of phagocytosis was calculated as ( # of bacteria with neutrophils from gentamicin-treated wells ) / ( # of bacteria from non-gentamicin-treated wells ) . High-bound tissue-treated 4HBX 96-well plates ( Fisher Scientific ) were used for all ROS assays . Plates were prepared as follows . For integrin activation , wells were coated with 15 μg/ml poly-RGD ( Sigma ) for 2 hr at 37°C or overnight at RT . For Fcγ receptor activation by IgG immune complexes ( IC ) , wells were coated with 20 μg/ml human serum albumin ( Sigma ) for 1 hr , washed twice with PBS , blocked with 10% FBS for 30 min , washed twice with PBS , and incubated with anti-human serum albumin ( Sigma ) at 1:400 dilution in 10% FBS for 1 hr at 37°C . Control wells were coated with 10% FBS for 30 min at 37°C . All wells were washed twice with PBS prior to use . For experiments with K . pneumoniae , wells were coated with 10% FBS for 30 min at 37°C and washed twice with PBS prior to use . For all ROS assays , 100 nM of PMA was used as positive control . To evaluate whether Skap2-/- neutrophils generated ROS after infection with another species of bacteria , a Y . pseudotuberculosis YPIII type 3 secretion mutant , ΔyscF was used ( Green et al . , 2016 ) . To detect RGD or IgG IC-stimulated ROS , superoxide detection by cytochrome C ( Sigma ) was performed as previously described ( Lowell et al . , 1996 ) with the following modifications . Cytochrome C was added to each well at a final concentration of 100 μM and 1 × 105 neutrophils were added to each well coated with either FBS or ligand . Absorbance at 490 nm and 550 nm were recorded at 37°C using Biotek Synergy HT . Superoxide concentration was calculated by ( OD550-OD490 ) x ( 2 . 11 × 104 M−1cm−1 ) , which is the extinction coefficient of cytochrome C ( Dahlgren et al . , 2007 ) . Total superoxide production was calculated by the sum of the area under the curves for the indicated time . To detect ROS production following K . pneumoniae exposure , isoluminol was performed as previously described ( Kobayashi et al . , 2016; Dahlgren et al . , 2007 ) with the following modifications . Neutrophils resuspended in HBSS+ , rested at RT for 30 min , were loaded with 50 μM isoluminol ( Sigma ) and 15 U/ml HRP ( Sigma ) , added to a 96-well plate at 1 × 105 cells/well , and incubated at 37°C for 10 min . K . pneumoniae ( grown as described in gentamicin protection assays ) were then added to wells at a MOI of 2 . 5 or 5 , and the plate was centrifuged at 500 x g for 3 min at 4°C . For inhibitor studies , following isoluminol and HRP loading , DIV neutrophils were pre-treated with 10 nM PP2 ( Selleck Chemical ) , 2 μM R406 ( Selleck Chemical ) , 1 μM Ibrutinib ( Selleck Chemical ) , 10 μM Go 6983 ( Abcam ) , 1 μM U-73122 ( Sigma ) , 1 μM U-73433 ( Sigma ) , or DMSO ( 1:10 , 000 final concentration , Sigma ) vehicle control for 10 min at 37°C with 5% CO2 . A BioTek Synergy HT late reader was used to detect chemiluminescence ( RLU ) . Total ROS ( total RLU ) production was calculated by the sum of the area under the curves for the indicated time in figure legends . DIV neutrophils ( 7 . 5 × 105 cells/well ) in HBSS+ were plated onto 96-well 4HBX plates coated with 10% FBS . K . pneumoniae was added at MOI 40 and spun at 250 x g for 2 min . Cells were incubated at 37°C in presence of 5% CO2 for 10 or 15 min , lysed in 1X Novex buffer ( 4X stock: 40% sucrose , 6 . 82% Tris-Base , 6 . 66% Tris-HCL , 8% SDS , 0 . 06% EDTA , 0 . 075% Bromophenol Blue , 2 . 5 mM NaVO4 , 100 mM DTT ) and 5–7 . 5 × 105 cells equivalents were resolved on 4–12% NuPAGE gel ( Invitrogen ) in MOPS buffer ( 10 . 5% MOPS , 6% Tris-Base , 5% SDS , 0 . 3% EDTA ) . Proteins were transferred to Immobilon-FL PVDF ( Millipore Sigma ) and subjected to western blot analysis using antibodies at a dilution of 1:500 unless otherwise indicated . Antibodies to phosphorylated proteins include Src Family-Y416 ( Cell Signaling Technology ) , Syk-Y352 ( Cell Signaling Technology ) and Pyk2-Y402 ( Cell Signaling Technology ) . All blots were also probed with RhoGDI ( Cell Signaling Technology ) at a dilution of 1:1000 as loading control . Secondary LI-COR goat anti-mouse IRDye 800 CW ( Cell Signaling Technology ) and goat anti-rabbit IRDye 800 CW ( Cell Signaling Technology ) were used at a dilution of 1:20 , 000 . The Odyssey CLx LI-COR system was used to develop the blots and IS Image Studio was used for analysis and quantification of bands . Blots were then stripped with 7M Guanine Hydrochloride ( Fisher Scientific ) by incubating at 56°C for 30 min with shaking , washed 3 times in 1x TBST ( 0 . 1% Tween-20 in 1x TBS ) , and reprobed with antibodies for total proteins against Syk and Pyk2 ( Cell Signaling Technology ) . The normalized protein level of pSFKs was calculated by taking the ratio of phosphorylated protein to RhoGDI loading control . The normalized protein levels of pSyk and pPyk2 were calculated by first taking the ratio of phosphorylated to total protein and then normalizing to the RhoGDI loading control . Normalized phospho-protein levels were used to calculate the fold change to unstimulated ( 0 min ) control within each group , or to unstimulated control of WT ( Supplementary file 1 ) . In addition , the relative induction of phosphorylation in unstimulated controls was determined by dividing the fold change of each experiment by the average of the three experiments . Fold change was log transformed and subjected to statistical analysis . Differences between the groups were evaluated by one-way ANOVA or two-way ANOVA with either Tukey’s or Sidak’s post-test , where appropriate ( GraphPad Software version 7 ) . Log-transformed values were used for determining significance in experiments measuring CFU .
Klebsiella pneumoniae is a respiratory , blood , liver , and bladder pathogen of significant clinical concern . We show that the adaptor protein , SKAP2 , is required for protection against K . pneumoniae ( ATCC 43816 ) pulmonary infections . Skap2-/- mice had 100-fold higher bacterial burden when compared to wild-type and burden was controlled by SKAP2 expression in innate immune cells . Skap2-/- neutrophils and monocytes were present in infected lungs , and the neutrophils degranulated normally in response to K . pneumoniae infection in mice; however , K . pneumoniae-stimulated reactive oxygen species ( ROS ) production in vitro was abolished . K . pneumoniae-induced neutrophil ROS response required the activity of SFKs , Syk , Btk , PLCγ2 , and PKC . The loss of SKAP2 significantly hindered the K . pneumoniae-induced phosphorylation of SFKs , Syk , and Pyk2 implicating SKAP2 as proximal to their activation in pathogen-signaling pathways . In conclusion , SKAP2-dependent signaling in neutrophils is essential for K . pneumoniae-activated ROS production and for promoting bacterial clearance during infection .
Klebsiella pneumoniae is a type of bacteria that can cause life-threatening infections – including pneumonia , blood stream infections , and urinary tract infections – in hospitalized patients . These infections can be difficult to treat because some K . pneumoniae are resistant to antibiotics . The bacteria are normally found in the human intestine , and they do not usually cause infections in healthy people . This implies that healthy people’s immune systems are better able to fend off K . pneumoniae infections; learning how could help scientists develop new ways to treat or prevent infections in hospitalized patients . In healthy people , a type of immune cell called neutrophils are the first line of defense against bacterial infections . Several different proteins are needed to activate neutrophils , including a protein called SKAP2 . But the role of this protein in fighting K . pneumoniae infections is not clear . To find out what role SKAP2 plays in the defense against pneumonia caused by K . pneumoniae , Nguyen et al . compared infections in mice with and without the protein . Mice lacking SKAP2 in their white blood cells had more bacteria in their lungs than normal mice . The experiments showed that neutrophils from mice with SKAP2 produce a burst of chemicals called “reactive oxygen species” , which can kill bacteria . But neutrophils without the protein do not . Without SKAP2 , several proteins that help produce reactive oxygen species do not work . Understanding the role of SKAP2 in fighting infections may help scientists better understand the immune system . This could help clinicians to treat conditions that cause it to be hyperactive or ineffective . More studies are needed to determine if SKAP2 works the same way in human neutrophils and if it works against all types of K . pneumoniae . If it does , then scientists might be able use this information to develop therapies that help the immune system fight infections .