NLRC4 suppresses IL-17A-mediated neutrophil-dependent host defense through upregulation of IL-18 and induction of necroptosis during Gram-positive pneumonia

Gram-positive pathogens, including Staphylococcus aureus cause necrotizing pneumonia. The central feature of S. aureus pneumonia is toxin-induced necroptosis of immune and resident cells, which impedes host defense. However, the role of the NLRC4 in the lung following S. aureus infection remains elusive. Here, we demonstrate that S. aureus activates the NLRC4 to drive necroptosis and IL-18 production, which impaired IL-17A-dependent neutrophil-mediated host susceptibility. In particular, Nlrc4−/− mice exhibit reduced necroptosis, enhanced neutrophil influx into the lungs, decreased bacterial burden, and improved host survival. Loss of NLRC4 signaling in both hematopoietic and non-hematopoietic cells contributes to the host protection against S. aureus pneumonia. Secretion of IL-17A by γδ T cells is essential for neutrophil recruitment into the lungs of Nlrc4−/− mice following infection. Moreover, treatment of wild-type mice with necroptosis inhibitors or genetic ablation of MLKL and IL-18 improves host defense against S. aureus infection, which is associated with increased IL-17A+ γδ T cells and neutrophils. Taken together, these novel findings reveal that S. aureus activates the NLRC4 to dampen IL-17A-dependent neutrophil accumulation through induction of necroptosis and IL-18. Thus, modulating the function of the NLRC4 may be an attractive therapeutic approach for treating S. aureus infections.


INTRODUCTION
cytosolic sensor for bacterial flagellin and type III secretion system (T3SS) 19,20 . However, Klebsiella pneumoniae, a non-flagellated bacterium lacking T3SS, and a flagellin-deficient strain of Pseudomonas aeruginosa have been shown to activate the NLRC4, suggesting its activation by other bacterial or endogenous host ligands 21,22 . Nonetheless, the role of NLRC4 in bacterial pneumonia caused by a Gram-positive organism still remains elusive 21,23 . Although recent studies linked NLRP3 and ASC to S. aureus toxin-induced necroptosis, the precise mechanism for this has not been elucidated either 7,18 . Furthermore, Nlrp3 −/− mice do not show a completely protective phenotype in S. aureus pneumonia indicating both NLRP3-dependent and independent mechanisms may be important in eliciting lung damage 13 . To this end, we investigated whether the activation of the NLRC4 is important in the pathogenesis of S. aureus pneumonia in a mouse model. We found that NLRC4-driven necroptosis and IL-18 suppress IL-17A signaling from γδ T cells leading to attenuated neutrophil recruitment, which is necessary to eradicate S. aureus from the lung.
Thus, these findings provide novel insight into how necroptosis and IL-18 dampens host defense in S. aureus pneumonia and suggests the inhibition of NLRC4 as a potential therapeutic approach for control of S. aureus infection in the lungs.

NLRC4 expression is enhanced in human and murine pneumonic lungs.
To determine whether NLRC4 expression is upregulated in human pneumonic lungs, we performed immunofluorescence staining of NLRC4 in lung sections from pneumonic patients and healthy controls. As virulence factors from S. aureus are known to target both myeloid and non-myeloid cells 8,10 , we assessed NLRC4 expression in myeloid cells (neutrophils, macrophages) and non-myeloid cells (epithelial cells) and found increased expression of NLRC4 in Lipocalin-2 + neutrophils, CD68 + macrophages, and CD326 + epithelial cells (Figure 1a). To explore if NLRC4 expression is increased during Grampositive pneumonia in mouse lungs, we performed immunofluorescence staining in lung sections at 24 hours post-S. aureus infection. We observed enhanced expression of NLRC4 in Ly6G + neutrophils, F4/80 + macrophages, and CD326 + epithelial cells in infected wildtype (WT) mouse lungs compared to that in control lungs ( Figure 1b). Collectively, our results indicate both myeloid and non-myeloid cells show up-regulation of NLRC4 during bacterial pneumonia.

NLRC4 deficiency contributes to host protection against S. aureus pneumonia.
To characterize the importance of NLRC4 in S. aureus pneumonia, WT and Nlrc4 −/− mice were infected intratracheally (i.t.) with a lethal inoculum of S. aureus (2×10 8 CFU/mouse) and survival was monitored up to 100 hours. Compared to WT counterparts, Nlrc4 −/− mice showed enhanced survival (Figure 2a). To determine if the enhanced survival in Nlrc4 −/− mice was due to increased bacterial clearance, WT and Nlrc4 −/− mice were infected i.t. with a sub-lethal inoculum of S. aureus (5×10 7 CFU/mouse). Compared to WT mice at 12 and 24 hours post infection (hpi), Nlrc4 −/− mice showed a diminished bacterial burden in the lung and bronchoalveolar lavage fluid (BALF) along with decreased dissemination in the spleen (Figures 2b-d). We next determined whether the enhanced bacterial clearance was due to augmented neutrophil accumulation in the lungs. Our results revealed that Nlrc4 −/− mice had increased numbers of total white blood cells, neutrophil, and macrophage recruited to the lungs (Figures 2e-g). Furthermore, unlike Nlrc4 −/− mice, WT mice had increased protein leakage, an indicator of lung permeability (Figure 2h). To assess if the detrimental role of NLRC4 activation is MRSA strain-specific, we infected WT and Nlrc4 −/− mice with a methicillin-susceptible S. aureus (MSSA) Newman strain. The protective phenotype seen in Nlrc4 −/− mice infected with S. aureus was also seen in pneumonia caused by the MSSA strain (Figures 2i, j).
S. aureus targets both myeloid and non-myeloid cells 7,10,11 . In Figure 1 a-b, we demonstrate increased expression of NLRC4 in both hematopoietic (myeloid cells) and nonhematopoietic (epithelial cells) cells in human and murine pneumonic lungs. Therefore, we next investigated the contribution of hematopoietic versus non-hematopoietic cells to host protection using bone marrow chimeras. In this context, lethally irradiated WT or Nlrc4 −/− mice were reconstituted with RBC-lysed bone marrow cells (4×10 6 /mouse) from donor WT or Nlrc4 −/− mice. Two months after reconstitution, chimeric mice were infected with S. aureus. Interestingly, the Nlrc4 −/− → Nlrc4 −/− , Nlrc4 −/− → WT but not WT → Nlrc4 −/− chimeric groups had decreased bacterial burden in lungs as compared with the WT → WT group (Figures 2k). However, the Nlrc4 −/− → Nlrc4 −/− , Nlrc4 −/− → WT, and WT → Nlrc4 −/− groups had reduced bacterial burden in BALF as compared to WT → WT group (Figures 2i). Taken together, our results suggest NLRC4 activation, in both hematopoietic and non-hematopoietic compartments, is detrimental, and their deficiency in mice displays robust protective phenotype in S. aureus pneumonia.

S. aureus activates NLRC4 through PKC-δ to induce IL-1β, IL-18, and necroptosis.
S. aureus α-hemolysin has been shown to activate NLPR3 in human and mouse monocytes 7 . Along the same line, to examine the virulence factors associated with NLRC4 activation, we used an isogenic α-hemolysin deficient S. aureus mutant strain, in human and murine cells. Direct evidence of increased NLRC4 expression in humans was obtained using lung lysates from pneumonic patients and comparing them to lysates from healthy individuals (Supplementary Figure 1a). In a similar manner, S. aureus activated NLRC4 in both human monocyte-like (THP-1) and neutrophil-like (HL-60) cells (Supplementary Figure 1a). To further characterize the role of NLRC4 in response to S. aureus infection, we used THP-1 and THP1-NLRC4 cells (stably overexpressing NLRC4). Compared to THP-1 cells, THP1-NLRC4 produced greater amounts of IL-1 β and IL-18 in response to S. aureus (Figure 3a Figure 1c). Recently, protein kinase C-δ (PKC-δ) mediated phosphorylation of NLRC4 was shown to be critical for inflammasome activation 24 . As anticipated, S. aureus-induced NLRC4 phosphorylation was dependent on PKC-δ in macrophages, as Rottlerin (PKC-δ inhibitor) reduced expression of phospho-NLRC4 ( Figure  3e). We also tested whether S. aureus-induced NLRC4 activation leads to caspase-1 processing and IL-1β production, key features of inflammasome activation. To this end, we Paudel  stimulated BMDMs from WT and Nlrc4 −/− mice with S. aureus, its isogenic αhemolysin deficient mutant, or purified αhemolysin toxin. As expected, the cleavage of pro-caspase-1 to its active (phospho-20) form was reduced in Nlrc4 −/− BMDMs, which was independent of the expression of αhemolysin by S. aureus (Figure 3f). This observation indicates S. aureus may expresses virulence factors other than α-hemolysin that may serve as activators of NLRC4-dependent caspase-1 processing. In addition, NLRC4 deficiency modestly reduced IL-1 β production in S. aureus-infected BMDMs, so as the pre-treatment with Rottlerin ( Supplementary Figure 1d, e). Furthermore, Nlrc4 −/− mice had significant reduction in key inflammasome-dependent cytokines, such as IL-1β and IL-18 (Figures 3g, h).
S. aureus has been shown to induce necroptosis in lungs 17 Therefore, in order to assess the role of NLRC4 in context of necroptosis, we performed cytotoxicity assay in THP-1 and BMDM following S. aureus infection. We detected increased cytotoxicity of THP1-NLRC4 and WT-BMDM compared to THP-1 and Nlrc4 −/− -BMDMs ( Figures S2a-c). It should be noted that IL-17A has been shown to be critical for neutrophil influx and bacterial clearance at the site of S. aureus infection 26 . Thus, we measured the level of IL-17A in BALF and found that IL-17A was increased in Nlrc4 −/− mice at 12 and 24 hpi (Figure 4c). To further implicate IL-17Amediated neutrophil recruitment in Nlrc4 −/− mice, administration of recombinant IL-17A in WT mice one-hpi increased neutrophil influx to a level comparable to Nlrc4 −/− mice ( Figure  4d). Further, ablation of Il17a or blockade with anti-IL-17A antibody in Nlrc4 −/− mice abrogated neutrophil influx, which was associated with an increased bacterial burden in BALF and lungs (Figures 4e-g). Since IL-17A is not directly involved in neutrophil recruitment, we explored whether enhanced recruitment may be due to increased level of CXC chemokines and neutrophil mobilization through IL-17A/G-CSF axis. Accordingly, Nlrc4 −/− mice also showed increased production of CXCL5, but not CXCL1 and CXCL2, at 12 hpi and higher trend of G-CSF at both 12 and 24 hpi (Supplementary Figures S2d-g). To examine if NLRC4 deficiency also alters the phagocytic function of neutrophils, we utilized a phagocytosis assay in bone marrow-derived neutrophils (BMDNs). To our surprise, WT-BMDNs show higher intracellular bacterial survival than Nlrc4 −/− -BMDNs following S. aureus infection ( Figure 4h). Further, Nlrc4 −/− BMDNs were also effective at particle uptake ( Figure 4i), suggesting NLRC4 deficiency improved both uptake and clearance in BMDN. Furthermore, the defect in uptake/phagocytosis of WT-BMDN was rescued by pre-treatment with IL-17A, which was associated with increased production of intracellular reactive oxygen species (ROS) (Figures 4i, j). Unlike in BMDNs, NLRC4 was dispensable for the phagocytic function of BMDMs (Supplementary Figure S3a). Moreover, deficiency of IL-17F, another IL-17 homodimeric cytokine complex that binds to the same receptors (IL-17RA or IL-17RC) 27 , did not alter neutrophil recruitment or bacterial clearance in S. aureus pneumonia (Figures 4k-n). Collectively, our data indicate that the increased level of IL-17A drives the influx and improves the phagocytic abilities of recruited neutrophil in Nlrc4 −/− mice.

NLRC4 deficiency augments IL-17A + γδ T cells and neutrophils to regulate neutrophil influx.
Although IL-17A is known as a signature cytokine for CD4 + T cells (Th17 subsets), emerging evidence has revealed that multiple cell types, such as γδ T cells, NK/NKT cells, neutrophils, innate lymphoid cells, CD8 + T cells, and colonic epithelial cells, also secrete IL-17A 27  mice, as there was decreased neutrophil influx and increased bacterial burden in the BALF of these mice at 24 hpi (Figures 5f, g). We next asked if genetic deficiency of NLRC4 alters the production of IL-17A from γδ T cells in addition to their recruitment to the lungs. To this end, we FACS sorted CD3 + γδ-TCR + cells from spleens of naïve mice and stimulated them with S. aureus in the presence or absence of IL-23 in vitro. Surprisingly, the γδ T cells from WT and Nlrc4 −/− mice did not show altered production of IL-17A following IL-23 stimulation (Figure 5h). To examine the possibility of increased γδ T cell proliferation in lungs of Nlrc4 −/− mice, we injected BrdU intraperitoneally before S. aureus infection and stained for BrdU positive γδ T cells. We found that NLRC4 deficiency did not regulate the proliferation of this cell type in the lungs (Figures 5i, j). Thus, these data indicate increased frequency of γδ T cells from early stage and neutrophil at late stage in Nlrc4 −/− mice produce IL-17A that is critical of neutrophil recruitment to the lungs in S. aureus pneumonia.

NLRC4-driven necroptosis and IL-18 suppress IL-17A-dependent neutrophil recruitment by limiting γδ T cell expansion.
We showed that S. aureus-induced NLRC4 activation results in the production of IL-1β, IL-18, and necroptosis in macrophages and lungs ( Figure 3 and Supplementary Fig 1). Necroptosis-mediated loss of immune cells in WT mice, but not in the Rip3 −/− mice, has been shown to worsen the outcome of S. aureus pneumonia 17 (Figures 81, m). Taken together, our results indicate that NLRC4-mediated necroptosis and IL-18 act synergistically, independent of IL-1β, to suppress the recruitment of γδ T cells, which dampens IL-17A-mediated antibacterial defense in S. aureus pneumonia.

Blockade of NLRC4 signaling or necroptosis improves host defense in S. aureus pneumonia.
Like other inflammasomes, activation of NLRC4 results in cleavage of caspase-1 leading to production of IL-1β and IL-18 during S. aureus infection (Figure 3). To determine if deficiency of caspase-1 (downstream of NLRC4) results in a similar outcome in S. aureus pneumonia, mice lacking expression of Casp1/11 or pre-treated with Ac-yvad-cmk (caspase-1 inhibitor) were infected with S. aureus. As compared to WT controls, mice lacking Casp1/11 or WT mice pre-treated with Ac-yvad-cmk demonstrate reduced bacterial burden in the BALF and lungs as well as improved survival (Supplementary Figures S6a-c).
To determine if blockade of necrosome components also had a similar effect in S. aureus pneumonia, we infected mice pre-treated with Nec-1 with S. aureus. As anticipated, blockade of RIP1 with Nec-1 enhanced neutrophil recruitment and subsequent bacterial clearance in the BALF, which was associated with improved host survival rates ( Supplementary Figures S6d-f). As S. aureus-induced NLRC4 activation was dependent on PKC-δ (Figures 3e), we investigated the role of PKC-δ inhibition in host defense. To this end, mice were pretreated with Rottlerin or DMSO at 12 hours prior to infection. As expected, Rottlerin pre-treated mice show reduced IL-1β production, increased neutrophil influx and bacterial clearance at 24 hpi ( Supplementary Figures S6g-i). Collectively, our results indicate that the inhibition of the NLRC4 signaling or necroptosis signaling cascades improves host defense against S. aureus infection.

DISCUSSION
The pathogenesis of Gram-positive (S. aureus) pneumonia is mediated by its several virulence factors, such as exotoxins and surface proteins. These virulence factors target different innate signaling molecules in infiltrating leukocytes and resident epithelial cells. Moreover, growing evidence indicates that S. aureus is able to exploit innate signaling pathways, as mice deficient in Adam10, Nlrp3, Nod2, Ifnr-1, and Tnfr expression showed augmented host defense against this pathogen [10][11][12][13][14] . Although toxin-induced necroptotic lung damage has been implicated in the pathogenesis of S. aureus pneumonia 17 , the precise mechanism underlying the subversion of host immunity is largely unknown. We report that NLRC4 activation leads to necroptotic cell death and IL-18 production, which in turn impedes host defense in S. aureus pneumonia. Furthermore, we demonstrate a novel mechanism by which NLRC4-driven necroptosis and IL-18 production suppress γδ T cellderived IL-17A-dependent neutrophil recruitment during S. aureus infection. Finally, we show that blockade or genetic deficiency in NLRC4 signaling cascade and necroptosis pathway improve host defense in the context of S. aureus pneumonia.
Pulmonary host defense regulated by the NLRC4 is protective against Gram-negative pathogens, such as K. pneumoniae 21 , L. pneumophila 29,30 and B. pseudomallei 31 , but deleterious against P. aeruginosa 23,32 . In contrast, our study reports NLRC4 activation is deleterious in the context of Gram-positive pathogens, as Nlrc4 −/− mice show improved host defense and survival. In accordance with this, activation of other NOD-like receptors has also been shown to be detrimental during S. aureus infection. For example, Nod2 −/− and Nlrp3 −/− mice are less susceptible to S. aureus pneumonia 12,13 . Because NLRP3 recognizes a broad range of pathogens, including S. aureus 33 , NLRP3 activation was thought to be critical for the pathogenesis of S. aureus pneumonia. However, Nlrp3 −/− mice do not have a remarkable phenotype in S. aureus pneumonia, suggesting involvement of an NLRP3independent host defense mechanism in the response to this pathogen 13 . We showed both myeloid and resident cells express NLRC4 during S. aureus pneumonia. Our bone marrow chimeras reveal that NLRC4 expression in both compartments is detrimental with regards to bacterial control. It is important to note that WT → Nlrc4 −/− group (hematopoietic) had defective bacterial control in the lungs compared to other chimeric groups. Since neutrophil are a major player of S. aureus clearance and Nlrc4 −/− neutrophils are more efficient in controlling bacterial burden, it is possible that NLRC4 expression in myeloid cells of WT → Nlrc4 −/− (only hematopoietic expression) group may have dampened phagocytic function of these cells.
Based on data from experiments in THP-1 cells and BMDMs, the NLRC4 was important for caspase-1 activity and IL-1β production in response to S. aureus infection. Our findings also indicate that α-hemolysin induces phosphorylation of the NLRC4, which is a PKC-δdependent process like in S. typhimurium-induced NLRC4 activation in macrophages 24 . However, α-hemolysin expression in S. aureus was dispensable for caspase-1 processing. In addition, pulmonary infection with K. pneumoniae or P. aeruginosa (non-flagellated strains) has been shown to activate the NLRC4 in vivo 21,22 , indicating that virulence factors other than flagellin can activate NLRC4. In this regard, recent reports related to the involvement of NLRC4 activation in obesity-associated tumors, in progression of melanoma tumors, and in diabetic nephropathy suggest endogenous host proteins may serve as signals for NLRC4 activation [34][35][36] . Therefore, it is possible that unidentified bacterial components of S. aureus or host-derived proteins may act as homologues to known NLRC4-activating bacterial signals.
A number of S. aureus toxins are known to activate necroptosis, a highly pro-inflammatory mode of cell death. S. aureus-derived pore-forming toxins like Hla, Leukocidin AB, and PSMs leading to cytotoxicity has been well characterized 7,8,37 . Further, necroptosis results in irreparable loss of alveolar macrophages and anti-inflammatory signals, impeding clearance of S. aureus 17 . In addition to reduced phosphorylation of MLKL in the lungs of Nlrc4 −/− mice, we also found that NLRC4 interacts with RIP3 suggesting a potential role of NLRC4 in necrosome formation. However, it remains unknown if NLRC4 activation also alters the K + efflux or contributes to membrane destabilization leading to cell lysis. Unlike Nlrp3 −/− , Nod2 −/− , or Rip3 −/− mice infected with S. aureus 12,13,17 , the central feature of protection in Nlrc4 −/− mice is their ability to recruit sufficient numbers of neutrophils to the lungs, which is impaired in WT mice. In Nlrc4 −/− mice, this occurs through the increased frequency of γδ T cells, which provide the IL-17A signal to drive neutrophil influx. Previous studies have shown that IL-17A stimulates granulopoesis in bone marrow and regulates expression of CXC chemokines in lungs to recruit neutrophils 38,39 . In our model, it appears that IL-17A may have contributed to neutrophil recruitment via increased CXCL5 and G-CSF production in Nlrc4 −/− mice. While numerous immune cells can produce IL-17A at different stages of infection, many studies have highlighted the importance of early IL-17A production by γδ T cells during infection with S. aureus 26,40,41 and other extracellular bacteria 39,42 . In addition to γδ T cells, the increased frequency of IL-17A+ neutrophils at 24 hours post-infection may explain that neutrophils are also involved in the recruitment through an autocrine loop. Interestingly, MLKL deficiency or blockade of necroptosis with Nec-1 or MLKL inhibitors improves bacterial clearance, which is also associated with an increased frequency of IL-17A + γδ T cells and neutrophil recruitment. In addition to necroptosis, NLRC4-induced IL-18 production was found to suppress the γδ T cell-IL-17A-axis, as IL-18 deficiency resulted in improved neutrophil recruitment and clearance of S. aureus. Similar to our results, IL-18 has also been shown to impede host defense in pneumonia and other disease models 23,28,43 . However, IL-1β, another NLRC4dependent cytokine, was surprisingly found to be dispensable for this pathway. Thus, these two seemingly disparate NLRC4-dependent mechanisms, the necroptosis and IL-18, work synergistically to impede neutrophil recruitment in S. aureus pneumonia. However, future studies will be required to determine how deficiency of necroptosis and IL-18 leads to the increased frequency of IL-17A producing γδ T cells as early as 6 hpi.
The precise mechanism through which PKC-δ-dependent NLRC4 phosphorylation occurs following Gram-positive (S. aureus) infection remains unclear. It should be noted that a wide range of pathogens or host-derived factors, along with environmental pollutants, can activate NLRP3 33 . Along the same lines, it is possible that NLRC4 may recognize host-derived ligands during bacteria-induced inflammation in addition to recognizing pathogens /or their components. For example, since S. aureus -derived pore forming toxins destabilize the membrane of cells, it is possible that endogenous inflammatory ligands are released and subsequently activates NLRC4. However, elucidation of the specific ligands and mechanisms responsible for NLRC4 activation during Gram-positive S. aureus infection of the lung will require future studies.

BALF collection, cell counts, and bacterial burden.
BALF and organs were collected as described earlier 21 . In brief, mice were sacrificed, the trachea was exposed and cannulated with 20-gauge catheter, and the lungs were flushed four times with 0.8 mL of PBS containing heparin/dextrose. Total and differential cell counts were performed on BALF using light microscopy utilizing Quik-Dip Stain. Lungs/spleen were excised, homogenized in PBS, and plated in serial dilutions on TSA plates to quantitate the bacterial burden in organs after overnight incubation at 37 °C.

Bone marrow chimeras.
Bone marrow transplantation was performed to create Nlrc4 −/− chimera mice wherein the Nlrc4 deficiency was confined to either the hematopoietic cells (Nlrc4 −/− → WT) or nonhematopoietic tissue (WT → Nlrc4 −/− as described in our previous publications 21, 39 . In brief, recipient mice were lethally irradiated with a single dose of 1000 rad. Each recipient mouse received a tail vein injection of marrow cells (4 ×10 6 /mouse) and was kept on 0.2% neomycin-sulfate drinking water for 2 weeks. Pneumonia was induced at 2 months after the reconstitution. Four groups were generated (WT → WT, WT → Nlrc4 −/− , Nlrc4 −/− → WT, and Nlrc4 −/− → Nlrc4 −/− ). Our routine procedure results in at least 75 -85% reconstitution of blood leukocytes from donor mice, as confirmed by flow cytometry on donor cells expressing GFP.

Statistical analysis.
Data are expressed as means ± standard error of mean (s.e.m.) unless otherwise stated. Prism 7.0a software (GraphPad Software Inc.) was used for statistical analyses. The unpaired t-test, Mann-Whitney U test (non-parametric), or one-way ANOVA (followed by Bonferroni's post hoc comparisons) were used to analyze differences between groups, as appropriate. Survival curves (Kaplan-Meier plot) were compared using log-rank tests. A P-value of *<0.05, **p<0.01, and ***p<0.001 was considered significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  The bacterial burden in (k) lung and (l) BALF was quantitated at 24 hpi. Each symbol represents a single mouse (b, c, d, j, k, l). Data from a representative experiment are shown. All experiments were performed three times, but survival and chimera study were performed twice. Statistical significance was determined by log-rank (a), Mann-Whitney (b, c, d, j), unpaired t-test (e-i), one-way ANOVA (followed by Bonferroni's post hoc comparisons) (k, l). *p<0.05; **p<0.01; ***p<0.001. BALF: bronchoalveolar lavage fluid, CFU: colony forming unit, WBC: white blood cell, nd: non-detectable.