Febrile Neutropenia INTERNATIONAL VETERINARY EMERGENCY AND CRITICAL CARE SYMPOSIUM 2017 Melissa A. Claus, DVM, DACVECC Murdoch University, Murdoch, WA, Australia Neutrophil Function Neutrophils are an important part of the innate immune system. They are one of the first immune cells to be recruited to a site of infection or inflammation. They move from circulation into the tissues by attaching first loosely and then tightly to receptors on activated endothelial cells. Once adhered, they move between or through endothelial cells and pericytes into the interstitial space. There, they become activated when their cellular transmembrane proteins, called pattern recognition receptors (PRRs), bind to pathogen- associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), molecules associated with invading pathogens or necrotic tissues, respectively. Once activated, they begin the process of degranulation, where intracytoplasmic granules fuse to the outer plasma membrane. This serves two purposes: firstly, it releases substances into the interstitial space that improve migration through the extracellular matrix, and secondly, it allows specific receptors to be expressed on the neutrophil outer membrane that enable migration, phagocytosis, and preparation for the deadly oxidative burst Neutrophils have three main ways of killing pathogens. Firstly, they degranulate to release destructive peptides and proteases into either the extracellular matrix or an intracytoplasmic phagosome. Secondly, they assemble a reactive oxygen species generator (NADPH oxidase complex) on the membrane of a phagosome or on the outer cell membrane, which causes an oxidative burst when activated by microorganisms, Thirdly, they form neutrophil extracellular traps (NETs). During this process, deoxyribonucleic acid, histones, and other nuclear material combine with destructive peptides and proteases from intracytoplasmic granules and are expelled from the cell into the extracellular space. This web of cytotoxic material ensnares and kills pathogens while at the same time containing destructive molecules to prevent damage to regional tissues. Formation of NETs is called NETosis and is a type of programmed cell death different from apoptosis and necrosis Neutrophil Production Granulocyte-colony stimulating factor (G-CSF) is the most important cytokine responsible for maintaining neutrophil homeostasis. This cytokine is secreted primarily by activated conventional dendritic cells, and it promotes differentiation of progenitor cells into neutrophils, increases proliferation, decreases time to maturation, and increases release of neutrophils from the bone marrow. Neutrophil production, or myelopoiesis, likely exists along a continuum controlled by titrated activation of conventional dendritic cells, secreting variable levels of G-CSF depending upon level of activation. Titration of activation is contingent upon the minute-to-minute variation in PRR signaling with exposure to PAMPs from commensal microflora or invading pathogens. This theory gained traction based on a study of germ-free mice living in a pathogen-free environment, The mice remained healthy, but had a sustained neutropenia at levels 10% of normal, coupled with low serum G-CSF levels, showing lack of stimulation to increase myelopoiesis. There are four main regions in which neutrophils are found, including the bone marrow, blood vessels, tissues, and marginated in microvasculature, Segmented neutrophils arise from progenitor cells in the bone marrow. Varieties of cytokines including G-CSF signal their release from the bone marrow, and they enter the circulating pool of neutrophils. These neutrophils move rapidly around the body in the larger vessels and make up the population sampled and counted in a complete blood count. The neutrophils that roll slowly along the endothelial cells of the microvasculature and stagnate in post-capillary venules make up the marginated pool of neutrophils. In dogs, about half of the neutrophils in circulation are in thecirculating pool and half are in the marginated pool. In cats, about a quarter of neutrophils are in the circulating pool, whereas three quarters are in the marginated pool. This different distribution requires that the definitions of neutropenia differ between the two species. In dogs, neutropenia is defined as <2900 cells/jiL. In cats, neutropenia is defined as <2000 cells/uL. Pathophysiology of Neutropenia Neutropenia compromises the function of the innate immune system and greatly increases the risk of sepsis. Invading opportunistic pathogens unchecked by the incapacitated innate immune system stimulate fever development, Febrile neutropenia has multiple etiologies and may arise whilst in hospital or may be the cause for presentation to the hospital. The three main mechanisms by which febrile neutropenia develop include increased utilization, immune-mediated destruction, and decreased egress from bone marrow. Increased Utlization Neutrophils are recruited to regions of infection or inflammation in response to circulating chemokines and cytokines that are generated by local activated dendritic cells and other neutrophils. The more extensive the infection or region of tissue inflammation, the higher the concentration of circulating cytokines and chemokines, Within the first few hours, excessive recruitment will deplete the circulating neutrophil pool. This drop lowers the concentration of chemokines in the bone marrow that retain mature neutrophils in the marrow. As a result, stored mature neutrophils egress into circulation. If the site of inflammation or infection is overwhelmingly large, the pool of stored neutrophils in the bone marrow may become depleted, resulting in persistent neutropenia. Complicating matters, bone marrow production of neutrophils may be decreased in sepsis. Astudy assessing septic mice found compromised ability of the bone marrow to replete the population of circulating neutrophils due to inhibited differentiation of hematopoietic progenitor cells into committed myeloid progenitor cells. Decreasing the differentiating capacity of the bone marrow maximally affects the neutrophil concentration, as this cell line has the shortest half-life of the blood cells, especially during a period of increased extravasation. Other studies have shown similar effects on myelopoiesis in the presence of the inflammatory cytokine IFN-y, as well as in murine models of sepsis and thermal injury. Immune-Mediated Destruction Similar to other immune-mediated diseases in dogs and cats, immune-mediated neutropenia is characterized as primary (idiopathic), or secondary to a trigger like neoplasia, medication, or infection. Destruction of neutrophils occurs when antibodies are produced against and then bind to neutrophil surface proteins, leading to phagocytosis by macrophages. Diagnosing this condition is difficult, as flow cytometry is required to detect the presence of anti-neutrophil antibodies in circulation, and this test is not commercially available. Instead, diagnosis is usually based on exclusion of all other causes of neutropenia in conjunction with response to therapy with immunosuppressive agents. Decreased Egress from the Bone Marrow Decreased circulating neutrophils may result from various disease processes directly affecting the bone marrow, There could be a depletion of granulocyte progenitor cells, which can arise due to infectious diseases, cytotoxic exposures, myelophthisis, or cyclic hematopoiesis, Alternatively, there may be normal to excessive quantities of granulocyte progenitor cells, yet ineffective myelopoiesis to produce mature neutrophils. This is known as dysmyelopoiesis and can arise due to infectious diseases, myelodysplasia, lithium administration in cats, acute myeloid leukemia, and trapped neutrophil syndrome of border collies. Depletion of Granulocyte Progenitor Cell Infection of hematopoietic progenitor cells with Parvovirus leads to apoptosis. Although all cells in the bone marrow become infected, neutrophils are the earliest and most severely affected due to their extremely short half-life of 6-10 hours. Neutrophil hatlife is likely even shorter in Parvovirus-infected dogs and cats due to increased extravasation and utilization in the gut, recruited because of invasion of intestinal microflora through the compromisedmucosal barrier, Neutropenia from depletion of progenitor cells has been seen with other infectious diseases as well, including rickettsial infections in dogs, and retroviral infections in cats. Many substances have been reported to cause neutropenia, including chemotherapy drugs, exposure to radiation, estrogens, and a handful of other drugs including antiepileptics, methimazole, antimicrobials, phenylbutazone, colchicine, and captopril. Chemotherapeutic agents and radiation both are designed to target and interfere with the cell cycle of rapidly dividing cells. Unfortunately, hematopoietic progenitor cells are part of the population targeted, which leads to progenitor apoptosis and severe neutropenia, Estrogens cause neutropenia indirectly. Exposure of thymic stromal cells to excessive levels of estrogens causes them to secrete myelopoiesis inhibitory factor, leading to depletion of progenitor cells. The mechanisms by which the other medications lead to neutropenia vary among different drugs, and are incompletely understood, Some possible causes include bone marrow necrosis or fibrosis, suppression of myelopoiesis, immune-mediated destruction, or a combination of these effects. Myelophthisis is the displacement of normal hematopoietic tissue by abnormal tissue, including neoplastic cells (typically round cell neoplasms), inflammatory cells, collagen (myelofibrosis), or bone (osteosclerosis). Most patients with these diseases have other cell lines affected, and almost all present with a non-regenerative anemia. Neutropenia develops with myelophthisis from a loss of granulocytic progenitor cells coupled with a loss of the nurturing bone marrow microenvironment that occurs with destruction of the stromal cells. Canine cyclic hematopoiesis (grey collie syndrome) is a genetic disorder in collies with a grey coat color. The disease is characterized by severe neutropenia occurring about every 2 weeks, with a drastic decline in the myeloid cell lines prior to neutropenia and myeloid hyperplasia prefacing neutrophil recovery. The genetic mutation is linked to low neutrophil elastase activity within the neutrophil, but it is unclear how this mutation causes myeloid depletion. It is postulated that neutrophil elastase must be involved with normal feedback inhibition during myelopoiesis. Ineffective Myelopoiesis Despite Normal to Excessive Quantities of Progenitor Cells Dysmyelopoiesis describes the presence of normal to increased quantities of dysplastic hematopoietic cells in the marrow coupled with leukopenia. Myelodysplastic syndrome and secondary dysmyelopoiesis are the two main acquired disease processes leading to this form of neutropenia. Myelodysplastic syndrome occurs with clonal expansion of a mutated hematopoietic progenitor cell. Daughter cells undergo apoptosis before they are released from the marrow. Cytologically, the bone marrow is hyperplastic with excessive blasts. This syndrome has been found in cats infected with feline leukemia virus and with acute myeloid leukemia. Secondary dysmyelopoiesis looks cytologically similar to myelodysplastic syndrome, except there is a normal number of blasts present. Secondary dysmyelopoiesis can occur with different diseases including immune-mediated hemolytic anemia, immune- mediated thrombocytopenia, and lymphoma. It can also be seen with the administration of some of the same drugs that can lead to neutropenia with hypoplastic bone marrow as described above (estrogen, phenobarbital, cephalosporins, chloramphenicol, and colchicine, as well as lithium in cats). A genetic disease in border collies leading to hyperplastic myelopoiesis with severe neutropenia is trapped neutrophil syndrome (TNS). Although the gene mutation that causes TNS has been identified, the underlying mechanism by which this mutation leads to decreased release of segmented neutrophils into circulation remains unknown, Clinical Presentation and Diagnostic Tests The underlying cause for neutropenia wil influence the clinical presentation. If infection precedes neutropenia, signs of sepsis together with a focus of suppurative inflammation and a degenerative left shift on complete blood count will usually be apparent. If neutropenia precedes infection, patients may have opportunistic infections with few clinical signs or abnormalities on diagnostic testing because of suppression of the normal inflammatoryresponse. It is important to note that fever will not always be present with neutropenia. Animals that have received fever-suppressing medications and animals in decompensated septic shock may be normothermic or hypothermic on presentation to the emergency room Diagnostic testing should be geared toward determining the underlying cause of neutropenia to allow for structuring of an appropriate treatment plan, Diagnostic tests to consider in a patient with neutropenia include blood cultures, urinalysis and culture, imaging of the thorax and abdomen, and bone marrow evaluation. Per the 2016 Surviving Sepsis Guidelines, two or more sets of aerobic and anaerobic blood cultures should be collected, ideally prior to administering antimicrobials. Positive blood cultures will provide a susceptibility profile to enable de-escalation of antimicrobial therapy. Severe neutropenia may preclude an active sediment on urinalysis, despite the presence of a urinary tract infection. Therefore, a urine culture is required to rule out an infection. Additionally, if an infection is present, the susceptibility profile will enable de-escalation of antimicrobials. Imaging of the thorax and abdomen using radiographs and ultrasound can help identify a septic focus. Imaging can also screen for other underlying diseases that could have led to neutropenia, Echocardiogram may be required to investigate the cardiac valves for vegetative lesions if the clinical picture is consistent, Bone marrow aspiration and/or a core biopsy may be required to determine the underlying cause of neutropenia, and this diagnostic test should be considered early if other blood cell ines are affected. Infiltrative neoplasia, myelofibrosis, osteosclerosis, and dysmyelopoiesis can only be diagnosed on a bone marrow sample. Immune-mediated neutropenia may be suspected with cytology consistent with maturation arrest. Finally, serial bone marrow cytologies may be helpful to. determine response to therapy and prognosis for recovery. Treatments and Supportive Care Cardiovascular resuscitation using intravenous crystalloids and vasopressors should be used as needed in neutropenic patients with septic shock. Additionally, if a septic focus is, identified, source control should occur as soon as possible. Patients with sepsis and septic shock are at risk of developing multiple organ dysfunction syndrome, They need to be carefully monitored and supported in an intensive care setting All febrile neutropenic patients require treatment with broad-spectrum antimicrobials, and treatment should begin as soon as possible. Antimicrobial choices should be based on suspected pathogen, suspected site of infection, and likelihood of infection with antimicrobial-resistant organisms. In febrile neutropenic people without septic shock, monotherapy with antipseudomonal B-lactams like ceftazidime or piperacillin-tazobactam is efficacious. Combination therapy using drugs from two different antimicrobial classes is recommended for people in septic shock. Antifungals should be administered if fungal infection is suspected. Appropriate antimicrobial stewardship requires de-escalation of antimicrobials to reflect susceptibility profiles. A medication to consider in dogs or cats with neutropenia is recombinant canine G-CSF. This medication increases maturation of progenitor cells and may improve function of circulating segmented neutrophils. It has been effectively used in both dogs and cats to increase myelopoiesis. In several dog studies, it increased recovery from neutropenia due to Parvovirus, chemotherapy agents, and cyclic hematopoiesis. Long-term safety studies in cats have not been performed, and there is some evidence of increased mortality in dogs administered this medication for parvoviral-induced neutropenia. Recombinant human G- CSF is also effective at increasing myelopoiesis in dogs and cats. However, use of this drug in dogs has been linked with chronic neutropenia due to development of anti-G-CSF antibodies, which cross-react to endogenous G-CSF. Use of either canine or human recombinant G-CSF is at the clinician's discretion. Use in dogs with parvoviral enteritis is not recommended Meticulous hand hygiene should be implemented when caring for all immunosuppressed patients. Hands should be washed and examination gloves should be worn prior to handling any indwelling devices including intravenous catheters, urinary catheters, feeding tubes, or tracheostomy tubes. Careful measures should be taken to keep these patients clean and dry to prevent opportunistic infections developing from their own commensal flora. At this time, insufficient evidence exists to recommend universal barrier nursing for these patients.References Bodey GP. Unusual presentations of infection in neutropenic patents nt J Antimicrob Agents, 2000;16:03-96, 2. Brown CD, Parnell NK, etal. 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Inflection points in sepsis bology: from lacal defense to systemic organ injury. Art J Physil Lung Cel Mol Physiol. 2012:303:1 395-1363, 10. Thomas CJ, Schroder K. Pattom recognition receptor function in neutrophils. Trends Immunol. 2013;34(7): 317— 328. SPEAKER INFORMATION (click the speaker's name to view other papers and abstracts submitted by this speaker) Melissa A, Claus, DVM, DACVECC Murdoch University Murdoch, WA, Australia URL: https:lwww.vin,comidoc/id=8172543