The host immune response to traumatic injury is complex with potentially serious consequences in certain situations. Research has shown that the immune system response after injury is dynamic with both arms of the immune system associated in the response. Building on existing work on trauma research, the exact mechanisms for how the immune system wreaks havoc in some trauma patients in the body’s attempt to restore homeostasis is not fully understood. An immunological cascade initiated after injury by certain innate immune cells has been indicated as the main catalyst for serious complications after trauma in SIRS (Systemic Inflammatory Response Syndrome). SIRS, a serious medical complication with inflammation throughout the body, is often associated with other conditions such as multiorgan failure leading to death. Early treatment and medical intervention are imperative in increasing the survival chances in those with severe complications. Despite the advances in medicine in the last few decades, therapeutic solutions to address these issues are still ongoing given the complex workings of the host immune system. This review aims to examine the immune response in various types of injuries, post injury impacts, current treatments, and ongoing research aimed at developing better therapeutic strategies.

Streptococcus pneumoniae (pneumococcus) is a pathogenic organism and the leading cause of bacterial pneumonia. Host-pathogen interaction is poorly understood, and factors that drive a more severe phenotype are unknown. During pneumococcal pneumonia, the innate immune system is critical for detection of invading pathogens. Macrophages are among the first responders to initiate a cellular host defense. This response is heterogeneous and the mechanisms behind the variability is unknown. An essential bacterial second messenger, cyclic di-AMP, has been shown to be secreted into the host cytosol and acts as a pathogen associate molecular pattern (PAMP) to initiate a robust type I interferon response. This type I interferon response occurs through the activation of cGAS and Stimulator of Interferon Genes (STING) pathway. Type I interferons are crucial in combatting viruses and there has been an emerging role for extracellular bacteria. The gram-positive bacterium, Streptococcus pneumoniae synthesizes cyclic di-AMP, and degradation occurs through two enzymes, phosphodiesterase 1 (pde1) and phosphodiesterase 2 (pde2). Using bacterial strains with a deletion of one (Δpde1 or Δpde2) or both phosphodiesterase enzymes (Δpde1Δpde2), we investigated the impact pneumococcal phosphodiesterases would have on macrophage responses. Along with type I interferon expression, the STING pathway can also activate the canonical NF-κB pathway, and all phosphodiesterase mutant strains elicited an increase in macrophage NF-κB activation compared to those infected with the wildtype bacterial strain. In contrast, it was revealed that only the phosphodiesterase 2 deleted pneumococci were able to incite an IFNβ response in macrophages that was not seen in other phosphodiesterase mutant strains (Δpde1 or Δpde1Δpde2). This excessive IFNβ response associated with faster macrophage cell death and depended upon macrophage uptake and phagolysosomal fusion. Overall, we conclude that pneumococcal phosphodiesterases appear to have differing responses on macrophages. Pneumococcal phosphodiesterase 2 mutation skews macrophage responses towards type I interferon, suggesting that deletion of phosphodiesterase 2 enzyme (and not the phosphodiesterase 1 enzyme) can activate selective arms of innate immune response. An improved understanding of pneumococcal phosphodiesterase 2 influences on bacterial physiology and host response could provide better insight into the host-pathogen responses.

The importance of TNF-TNFR signaling in immunity against M. tuberculosis has been established. The aims of this study were to characterize the functions of membrane-bound TNF (Tm-TNF) and soluble TNF (solTNF) and to investigate the role of membrane-bound p55TNFR signaling as well as the in vivo significance of TNFR shedding in host immune responses during infection with M. tuberculosis H37Rv. To address this, mice expressing only the membrane-bound TNF or membrane-bound p55TNFR were exposed to a low dose of M. tuberculosis H37Rv by aerosol inhalation infection. The results presented in this dissertation illustrate that Tm-TNF mice were able to control acute M. tuberculosis infection but succumbed to chronic exposure to M. tuberculosis with pneumonia. We demonstrate that Tm-TNF mice displayed heightened pulmonary macrophage activation reflected by enhanced cell surface expression of MHC-II, CD80 and CD86 as well as enlargement of granulomas. Furthermore, our results show that solTNF has a regulatory function that modulates the magnitude of Th1 immune responses during acute and chronic stages of the infection. The evaluation of the functions of Tm-TNF and solTNF in host immune function in the presence of an established mycobacteria-specific immune response was carried out using a 'drug-based' M. tuberculosis reactivation model. Here, mice that were challenged with a low dose of M. tuberculosis were exposed to INH-RIF treatment for six weeks in drinking water, after which therapy was withdrawn and immune responses during reactivation were analyzed. Our results demonstrate that complete absence of TNF resulted in host susceptibility to recrudescence tuberculosis in the presence of a mycobacteria-specific immune response. TNF deficient mice were unable to suppress bacilli growth and formed diffused granulomas and succumbed early to reappearing tuberculosis compared to WT mice. By contrast, we show that Tm-TNF was sufficient for containment of reappearing mycobacterial growth and sustaining immune pressure in a manner comparable to WT control mice. xii Lastly, the analysis of host immune responses in mice expressing a non-sheddable p55TNFR revealed that persistent p55TNFR cell surface expression does not afford better protection to low dose M. tuberculosis infection. However, we observed a transient elevation in the frequency of pulmonary CD11b+/MHC-II+ cells in mice expressing a non-sheddable p55TNFR relative to WT mice as well as reduced cell surface expression of CD44 on CD4+ T cells. We also found that pulmonary IL-12p70 and TNF concentrations were elevated whereas IFNγ levels were reduced in mice expressing a non-sheddable p55TNFR relative to WT mice. Furthermore, data presented here describe the in vivo functional significance of p75TNFR shedding. We demonstrate using a double mutant mouse strain that in the absence of p75TNFR, mice expressing a non-sheddable p55TNFR display enhanced ability to control M. tuberculosis infection.

Ulcerative colitis (UC) is a heterogeneous inflammatory bowel disease (IBD) associated with chronic inflammation of the gastrointestinal tract. Characterized by genetic and immunological abnormalities, UC has overly aggressive T-cell responses to commensal bacteria eventually leading to disease pathology. UC is distinguished from Crohn's disease, another form of IBD, in that it is driven by a T helper type 2 (Th2) immune response. Oxazolone-induced colitis is a mouse model resembling UC presenting with inflammation limited to the distal colon and mixed neutrophil/lymphocyte infiltration in the superficial layer of the mucosa. The Th2 cytokines interleukin (IL)-4 and IL-13 are associated with the onset of oxazolone colitis and both signal through a common IL-4 receptor-alpha chain (IL-4R +-). Neutralizing these cytokines prevents or ameliorates disease significantly, while neutralizing IL-12 exacerbates disease symptoms. As many aspects of the mechanisms involving Th2 cytokines in colitis remain undefined, the aim of this study was to investigate the role of IL-4 and IL-13 and the receptors through which they signal in oxazolone-induced colitis. Previous studies have highlighted a role for IL-4 and IL-13 in mediating oxazolone colitis. We show that while IL-13-deficient BALB/c mice were protected from disease onset, IL-4R +- deficient BALB/c mice developed exacerbated disease symptoms.

Bibliography: leaves 125-150.

Includes bibliographical references (p. 117-127). / The fish-parasitizing nematode Anisakis pegreffii induces gastrointestinal disease and allergy when ingested by humans, and can cause occupational allergy in seafood processing workers. The present study examines immune and allergic responses to A. pegreffii in wildtype and gene deficient mice, with special focus on interleukin(IL)-4, IL-13, and the IL-4 receptor alpha (IL-4Rα). Experimental murine models of Anisakis infection, Anisakis-induced anaphylaxis and Anisakis-induced dermatitis were established in order to gain insight into the immune responses generated against Anisakis and unravel mechanisms of allergic disease.

During the last decade, cholinergic signaling via acetylcholine and its receptors has emerged as an important regulator of immunity. Acetylcholine binds to and signals through two types of receptors; nicotinic and muscarinic receptors. Studies have shown that signaling through nicotinic receptors, particularly the α7 subtype on macrophages has potent anti-inflammatory effects. However, the role for muscarinic receptor has not yet been conclusively characterized. In this study, we demonstrate that M3 muscarinic receptor subtype is required for optimal protective immunity to two pathogens; the nematode Nippostrongylus brasiliensis and the bacterium Salmonella enterica sp. Tyhpimurium. M3R deficient mice (M3R-/-) were susceptible to infection with N.brasiliensis with decreased production of the protective cytokine IL-13. Furthermore, stimulation of lymphocytes with muscarinic agonists enhanced TH2 cytokine production in an M3R dependent manner.

Bibliography leaves 60-70. / Tuberculosis remains to be a leading infectious cause of death worldwide. This is in spite of the BCG vaccine against tuberculosis that has been in use for over 80 years as well as several chemotherapies. Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen targeting host cells, predominantly macrophages, to establish an infection. Endothelial cells form a barrier that has to be crossed by the bacilli in the establishment, and subsequent dissemination of mycobacterial infection. The present study was undertaken to investigate the phagocytosis of mycobacteria by endothelial cells and macrophages and the subsequent activation of these host cells after infection with the bacilli. Endothelial cells, obtained from the human umbilical vein (HUVEC) were infected with BCG-GFP under various conditions and uptake determined by means of flow cytometry. Endothelial cells phagocytised mycobacteria in a dose- and a time-dependant manner. Exposure of mycobacteria to serum opsonins enhanced the uptake of the bacilli by endothelial cells. However, heat killing of mycobacteria inhibited its uptake by endothelial cells. Data from the fluorescent microscope showed the association of BCG-GFP signal with endothelial cells detected on the FACS caliber. Analysis by confocal microscopy confirmed internalisation of endothelial cells by mycobacteria. Endothelial cells were further investigated for an acquired phenotype following infection with mycobacteria. CD31, a marker for endothelial cells, was neither down regulated nor up regulated. However, ICAM-1 expression, one of the adhesion molecules was down regulated upon infection of endothelial cells with mycobacteria. No TNF-α and IL-6 were detected in culture supernatants of infected endothelial cells. Macrophages obtained from murine bone marrow, phagocytosed mycobacteria in both a dose- and a time-dependant manner. Unlike endothelial cells, heat killing of mycobacteria did not obliterate their uptake by macrophages. However, macrophages preferentially phagocytosed viable mycobacteria in a 3h infection period, but not in an 18h period. Macrophages from the Mac-l mouse strain, lacking a phagocytic receptor 3 (CR3), were included in this study. The uptake of pathogenic mycobacteria, H37Rv by macrophages from Mac-1, was reduced in cell cultures infected for 4 hours but not those infected at 1 and 2 hours. Similarly, reduced uptake of avirulent mycobacteria strains H3 7Ra and BCG in the absence of CR3 was pronounced in cell cultures infected for longer periods. The activation state macrophages acquire after infection with mycobacteria was investigated with respect to the expression of MHC glycoproteins, and secretion of IL-10 and IL-12. The activation state of macrophages with respect to these parameters studied is critical in the interaction with T-lymphocytes, for subsequent containment of mycobacteria infection. Production of IL-12, a critical Th-1 cytokine, was proportional to MOI, and enhanced by viability of pathogenic mycobacteria. Furthermore, prior exposure of mycobacteria to serum opsonins inhibited the secretion of IL-12, while exposure of the bacilli to bronchoalveolar factors greatly enhanced it. IL-10 production by infected macrophages was on the other hand inhibited by prior exposure of mycobacteria to both serum and bronchoalveolar fluid factors. Macrophages consitutively expressed MHC I. After infection with pathogenic mycobacteria, cells positive for MHC I, were hardly detected in macrophage cultures infected with mycobacteria that had been exposed to fresh serum and bronchoalveolar fluid opsonins. MHC II on the other hand, was not consitutively expressed on macrophages. Following infection with pathogenic mycobacteria, the highest percentage of cells positive for the antibody against MHC II were observed in macrophage cultures infected both without any opsonin and in the presence of bronchoalveolar fluid factors. - In conclusion, the present study demonstrates that endothelial cells bind and internalise mycobacteria. That they get activated as evidenced in the down-regulation of ICAM-1 following infection with mycobacteria. Thus endothelial cells may not just be a passive, physical barrier but host cells that may have an active role in mycobacterial infection. Macrophages in comparison to endothelial cells were more effective in phagocytosis of mycobacteria in a time- and dose-dependant manner, differentiating themselves as professional phagocytes in the internalisation of heat-killed bacteria where the endothelial cells lacked the ability for the uptake of mycobacteria.

Introduction Sexual transmission of HIV-1 accounts for more than 80% of all the transmissions globally. After transmission, approximately 80% of the newly disseminated infections can be traced to a single variant, which comes from the minor HIV-1 population within the transmitting donor. This has led to the widely accepted idea of an HIV-1 transmission bottleneck. The nature of this bottleneck is not fully understood. Many studies working on understanding the nature of the transmitted virus have reported discordant traits of transmitted/founder viruses compared to viral isolates from chronically infected individuals. Such studies therefore lacked analysis of the intermediate step between these two populations: HIV-1 from the genital tract of the donors: where viruses are on their way to transmission to a new recipient. Importantly, numerous prior studies have shown that there is compartmentalization of HIV-1 populations between the general circulation and the genital tract, raising the possibility that the genital tract is an important selective environment. Collectively, prior studies of genital tract compartmentalization in males detected compartmentalization in about half of the donors studied, although by using techniques with limited depth of sampling than that employed in our study. The virus that establishes disseminated infection in a new recipient is selected. However, the extent to which this selection occurs before, during or after crossing the mucosal surfaces of the recipient is less clear – the period during which transmission selection could extend far back to the donor, and the donor’s genital tract. In other words, it is not clear as to what extent the transmission bottleneck occurs during compartmentalization of viral populations in the genital tract tissues. The design of an effective vaccine and other intervention strategies will rely upon the understanding the nature of the transmitted virus as this is the virus that must be targeted. This thesis Compartmentalization of minor variants cannot be tracked by techniques previously used to describe compartmentalization between the genital tract and the blood circulation. We therefore used deep sequencing-based techniques to further understand compartmentalization of viral populations between blood and the male genital tract. In addition, we tested the sensitivity of variants to a range of entry and other inhibitors in order to explore possible changes in function that may arise when viral variants grow in the shifted selective milieu of the genital tract. We further hypothesized that this change of selective milieu as HIV-1 moves from blood into the genital tract may lead to viral variants in semen that are sensitive to autologous neutralization because such sensitive variants may be able to grow in the genital tract, which is presumably partially or completely shielded from antibodies. Because the viral populations in semen comes from a site that may be relatively protected from antibodies, they may be permitted to evolve differently in the relative absence of antibody pressure. It is possible that evolution of the virus within the genital tract is a significant part of the change the virus undergoes on its way to establishment of a new disseminated infection in the new recipient. We considered this possibility because even some small molecules like those of some antiretroviral drugs do not penetrate the genital tract effectively under some circumstances, raising the possibility that antibodies might not always penetrate in all areas of the genital tract. This thesis had three objectives: 1. To evaluate HIV-1 compartmentalization in blood and the male genital tract using next generation sequencing to understand the nature of viral populations in these anatomical sites in greater detail. 2. To identify the differences in sensitivity of blood and semen variants to entry inhibitors to obtain information about differences in function between HIV-1 populations in blood vs the male genital tract. 3. To compare neutralization sensitivities of viral variants compartmentalized in blood and semen by testing their sensitivity to neutralization by autologous antibodies. As a control, we measured sensitivity to a pool of clade-matched heterologous sera to determine if any observed difference was due to global changes in neutralization sensitivity. Methods Study participants Forty-four HIV-1 seropositive men were recruited and then requested to donate blood and semen samples at ANOVA Health’s Ivan Toms clinics at Woodstock and Green Point or through their mobile clinic in Khayelitsha, all in Cape Town, South Africa. Viral loads from blood and semen and CD4+ T cell counts from blood were measured. HIV-1 was enriched from semen using a Nycodenz gradient, and then concentrated using ultracentrifugation. Chapter 2: HIV-1 Compartmentalization in blood and semen Next generation sequencing on Illumina paired-end Miseq platform was performed. To our knowledge there is no published study that has used this technique to study male genital tract HIV-1 variants in chronically infected male donors, although there is one that does so for acutely infected donors. We argue here that this is a superior method of sampling populations in blood and the male genital tract. In particular, it allowed us to more accurately track minor populations within each compartment. Additionally, the use of PrimerID approach allowed us to more clearly identify clonal amplification events in the HIV-1 populations. Sequencing was performed on either the V3 or C3-V5 region of the HIV-1 envelope gene from paired blood and semen samples from 11 donors. To evaluate compartmentalization, populations from blood and semen were compared using three standard techniques, Slatkin Maddison Test (SMT), Wrights measure of population subdivision (FST) and nearest neighbour statistic (Snn). Clonal amplification and results of modelling a lower depth of sampling are also presented. Chapter 3: Sensitivity of blood and semen variants to entry inhibitors and changes in function From three subjects who exhibited the highest extent of compartmentalization, full-length envelope clones derived from semen and blood RNA were made using limiting dilution PCR (single genome amplification), which provided the advantage of minimizing PCR-based artificial recombination. A high fidelity Taq polymerase was also used to minimize base-substitution errors. An average of 10 clones were isolated per compartment. Pseudoviruses were then constructed from the full-length envelope clones from blood and semen. The sensitivities of these pseudoviruses were tested against HIV-1 entry inhibitors; Maraviroc, PSCRANTES, enfurvirtide (T-20) and JM2987. Maraviroc and PSC-RANTES are CCR5 inhibitors while JM2987 is a CXCR4 inhibitor. Enfurvirtide (T-20) is a fusion inhibitor blocking the virus from entering cells. The full-length clones used to make the pseudoviruses were also sequenced and genomic variations in variable loop characteristics (length and number of potential glycosylation sites) between blood and semen compared. Chapter 4: Sensitivity of blood and semen variants to autologous and heterologous antibodies To study the differences in sensitivity of blood and semen variants to antibodies, pseudoviruses cloned from semen RNA and blood RNA (above) were tested for their sensitivities to donor antibodies collected at the same time the samples were collected or to a pool of HIV-1 subtype C sera. Results Objective 1: Viral compartmentalization via next generation sequencing HIV-1 populations were compartmentalized in all the 11 donors studied but to varying extents. Donor SVB043 had the most compartmentalized viral populations between blood and the male genital compartment using all the three measures of compartmentalization. Further analysis of the phylogenetic trees revealed that some clusters contained either purely blood or semen sequences, even in trees generated from analysis of donors with weakest compartmentalization. This might explain the viral compartmentalization signal in these weakly compartmentalized donors. To mimic reduced sampling depth, subsampling of the Illumina Miseq data with a small number of sequences was done. This analysis revealed that viral compartmentalization between blood and male genital tract would likely (>50% estimated likelihood) have been detected in only 5/11 (45%) of the donors, a proportion which is very similar to the aggregate proportion from previous studies that had used single genome amplification (SGA) analysis. This means that the difference in detecting HIV-1 compartmentalization in this thesis vs previous studies can be explained by the depth of sequencing achieved here and that there is no evidence that the dynamics of the viral populations studied in this thesis were different from those previously studied. In addition, the most recent common ancestor of semen variants was mostly located in blood, indicating the male genital tract was seeded by incoming variants from blood. Clonal amplification was also observed in all the 11 study participants and it was a characteristic of variants from blood and the male genital tract and its frequency did not obviously correlate with the severity of compartmentalization. In sum, blood and male genital tract HIV-1 compartmentalization and clonal amplification is present in most or all HIV-1 infected males but was not detected in all individuals in previous studies when using techniques with lower depth of sampling. Objective 2: Sensitivities of blood and semen variants to entry inhibitors and variable loop characteristics Viral variants from the most compartmentalized donors had variations in sensitivities to entry inhibitors; although the direction of the difference was inconsistent. Donor SVB043 who had the most severely compartmentalized viral populations between blood and semen, had semen viruses that were 1.67 (95%CI 1.08 – 2.56) times more resistant to maraviroc (p=0.024) while SVB008 which was the second most compartmentalized donor, had semen isolates that were 4.8 (95%CI 2.76 – 8.28) times more sensitive to inhibition by maraviroc (p < 0.0001). The meaning of this discrepancy is not entirely clear. It could mean that trait(s) that are selected for in genital tract variants over blood circulation variants are linked to the CCR5 binding region, and that the linked CCR5 genotype was carried along with the selected trait(s). There were no differences in sensitivity to maraviroc between blood and semen clones for donor SVB049 (p=0.847); although this donor on further investigation was found to have functional levels of efavirenz in his blood (3µg/ml, which were within the therapeutic range of 1-4µg/ml) indicating that he was likely on antiretroviral therapy (ART). This was not known to the clinic staff at the clinic at which he was known to receive care and was recruited to this study. The direction of sensitivities to PSC_RANTES (another CCR5 inhibitor) was concordant to that observed for maraviroc for donors SVB008 and SVB049 but not for donor SVB043 where semen variants were 1.67 (95%CI 1.08 – 2.56) times less sensitive than blood variants to maraviroc, with no detected difference in sensitivity to PSC_RANTES (p = 0.783). This discrepancy for donor SVB043 probably reflects the difference in mode of action between Maraviroc and PSC_RANTES. The change in envelope conformation over movement from blood into the genital tract presumably affected the maraviroc binding site and not PSC_RANTES. All the clones from blood and semen for the three most viral compartmentalized donors were resistant to CXCR4 inhibitor suggesting that they were all R5 tropic viruses. There were no differences in sensitivities of blood and semen viruses to fusion inhibitor T-20. The findings here suggest a changed viral envelope conformation/structure for the viruses in the male genital tract. The discordance suggests that the selected trait over movement of virus from blood into genital tract is linked or close to CCR5 binding site but itself does not involve binding to CCR5 coreceptor. Differences in length and number of glycosylation sites were found between variants from blood and those from the genital tract but the direction of the difference was also inconsistent. Donor SVB043 who had the most compartmentalized blood and seminal variants had semen variants that had longer and more glycosylated envelopes. Donor SVB008 who had the second most compartmentalized blood and semen variants had no difference in variable loop length, but semen variants were less glycosylated. This therefore shows that selection for some of the previous reported traits of acute viral isolates may have started in the genital tract in a subset of the donors. Objective 3: Sensitivities of blood and semen variants to autologous and heterologous antibodies Viral populations compartmentalized in blood and the male genital compartment displayed a range of sensitivities to autologous and heterologous neutralization. Donor SVB043 who had the most compartmentalized viral populations between blood and the genital tract; had semen clones that were 1.75 (95%CI 1.11-2.78) times more sensitive to autologous neutralization compared to blood clones (p = 0.018). In contrast, donors SVB008 and SVB049 who exhibited substantial compartmentalization, but to a lesser extent than that found in donor SVB043, showed no differences in sensitivities of blood and semen variants to autologous serum. Neutralization sensitivity to a pool of heterologous subtypematched sera revealed no differences in sensitivities between clones from blood and semen for donors SVB043 and SVB008. Interpretation of results from donor SVB049 are clouded by the donor’s ART use. Overall, these results suggest that, in some individuals, a shift in selective milieu of the genital tract virus occurs. This is presumably due to partial or complete shielding of the genital tract tissue from circulating antibodies, and this shielding shape the populations of HIV-1 variants available for transmission from some but not all individuals. Overall conclusions Our data add to the existing knowledge of existence of distinct viral populations between blood and the male genital tract of chronically HIV-1 infected donors. Importantly, and for the first time, we present evidence that HIV-1 compartmentalization between blood and the male genital tract is present in most or all donors, and that some clones are severely compartmentalized even in donors who exhibit very mild compartmentalization. It appears that viral compartmentalization and clonal amplification in these anatomical sites may be present in most individuals but remained undetected in some individuals in previous studies due to the lower depth of sampling applied. We observed a discordance in entry inhibitor sensitivities and variable loop characteristics between blood vs semen variants among different donors. This may suggest a changed envelope conformation over importation of virus from blood into the genital tract. This change seems to be near or linked to the co-receptor binding site but does not appear to directly involve the co-receptor binding tested in this thesis. This interpretation also may explain the discordance in viral characteristics for the virus establishing infection reported in other studies. This thesis also shows that some of these traits of the transmitted/founder virus relating to neutralization sensitivity, sensitivity to entry inhibitors and variable loop characteristics may originate in and/or be enhanced by transition through the genital tract on the way to becoming a founder virus. These results are important in understanding how the populations in the genital compartment are selected, giving rise to the population of HIV-1 that is available for transmission to a new individual. An understanding of the dynamics of HIV-1 populations prior to and during transmission is important for vaccine design and other intervention strategies.

Includes bibliographical references (leaves 147-157). / Clecsf8 is a poorly characterised member of the "Dectin-2 cluster" and was originally thought to be expressed exclusively by macrophages. In this study it was demonstrated that Clecsf8 is primarily expressed by peripheral blood neutrophils and monocytes.